Neuroscience research very frequently requires animal sacrifice and microscopic examination of the brain. Certainly, stereotaxic surgery is always followed by histological examination, if only to confirm the correct placement of the probe. In nearly every case, unless live tissue is required, the animal is sacrificed by washing out the blood and running fixative through the vascular system, a technique called perfusion. Sacrifice Perfusion should not be confused with organ culture perfusion, which requires running warmed, oxygenated fluid through tissue to keep it alive.
Ubiquitous in animal research
Perfusion as a means of getting fixative into tissues as rapidly as possible was described by Palay et. al. in 19626. The vascular system is an open channel giving access to every cell in the body within seconds. Cells began a process of breakdown by autolysis almost as soon as a state of anoxia is present, so the more rapidly the fixative is at every cell, the more uniform the resulting tissue quality throughout each organ. Formaldehyde (the most commonly preferred fixative) penetrates immersed tissue at a very slow rate, about 18 mm/ 25hours, depending on the tissue2. Thus, getting formaldehyde to every cell in the body within seconds has obvious advantages for tissue quality for whole organ work.
Since Palay, sacrifice perfusion has become almost ubiquitous in animal research labs, and profoundly influences how the tissues stain and look under the microscope. In spite of that universal acceptance, there has been no commercial apparatus with a considered manual. Each lab cobbles together its own apparatus, with limited understanding, and rarely mentions in the Methods section of journal articles the details of what was done.
Gravity versus peristaltic pumps
Palay et al6 used gravity pressure to drive a prewash of physiological saline, followed by the fixative, through the vascular channel. This method was apparently employed in most labs for some time. However, tissue perfused in this manner was usually left with a reddish hue of blood cells remaining in the tissue, sometimes some soft areas of tissue, and shrinkage of soft tissues. Red blood cells autofluoresce, and interfere with the common cell labeling reactions such as HRP. Still, the resulting tissue was much more uniform and suitable for research investigations than tissue immersed in fixative with all blood remaining.
Better results as to blood washout and uniformity of tissue were obtained by some labs using a peristaltic pump to drive the fluids, and that may be the more common method at present. Methods sections rarely report which method was used. A peristaltic pump controls flow rate, not pressure. Pump settings are determined by previous experience, there no obvious ways to calculate the correct flow rate.
Cardiovascular resistance varies
The laws of physics have it that for any fluid flow situation, flow rate equals the pressure divided by the resistance. If two variables are set, the third is determined by the equation. Cardiovascular resistance varies widely between species, strains genders, animal sizes, exercise state, and previous history. There are no algorithms to help predict what the cardiovascular resistance of a given animal will be, or hence what flow rate should be used to maintain a suitable pressure to open up capillaries. Pressure may vary widely between individuals when flow rate is controlled. The flow rate is invariably decided experientially, and seldom adjusted for differences between individuals or groups of animals. If a mouse and a rat were to be perfused at the same flow rate, whatever flow rate was chosen, one would be a very poor result. There would similarly be noticeable differences in quality of perfusion for younger vs. older rats, or a male and female rat of the same age.
In contrast, within limits, all mammals have the same blood pressure5. A pressure such as 200 mm of Hg would be above resting physiological levels in any mammal, attainable by intense exercise or excitement. A pressure of 300 mm Hg would be well above any physiologically attainable blood pressure in any mammal. Neither would cause harm if maintained for a very short period in an otherwise healthy animal. A pressure of 300 mm Hg would reliably wash out all the blood from every capillary, without damaging anything, in any mammal, even neonatal rats. Of course, the flow rates resulting would be dramatically different for different species and sizes of animal. What accounts for the inferior results using gravity flow, which is also a uniform pressure source?
The answer is in the math. One mm Hg equals 13.6 mm H20. A pressure of 300 mm Hg would require that the source prewash and fixative bottles be 4.08 meters above the animal, not possible given ceiling height in most labs. Even a physiological (systolic) pressure of 120 mm Hg would require the source to be 1.63 meters above the animal, still not possible if the animal is at sink height, and certainly not convenient. In practice, gravity perfusions are done at a fraction of physiological pressure, and all capillaries don’t clear. Where a capillary is blocked by red blood cells, fixative does not reach tissue areas served by that capillary as rapidly as other areas. Uniformity of fixation and tissue firmness suffers accordingly. Controlling pressure is the preferred procedure, but requires more pressure than can be obtained through gravity in a standard laboratory.
A drawback of perfusion is that soft tissue is usually shrunken and warped after common perfusion procedures. Cragg (1980)1 offered a hypothesis, and a procedure to avoid the shrinkage. Every cell has a variety of ion pump proteins on the cell surface. Most important is the sodium pump. Sodium continuously leaks into the cell, and is pumped out by an energy using process to maintain about a 10:1 outside to inside sodium concentration. Anything that interferes with cell energy metabolism (certainly including cold anoxic prewash solution) promptly shuts down the ion pumps. In that event, sodium moves from the extracellular space into the cell. Water must move in as well to maintain tonicity. Extracellular fluid moves into the cell, and the cell swells up to occupy the extracellular space. The absence of extracellular space in perfused tissue is a well known electron microscopy finding, but other methods have proven that such space exists7. At the end of perfusion, the membranes are more fully permeable, and the fluid leaves the cells into the vascular space. The extracellular space does not re-inflate, so the organ collapses inward by that amount. Ventricles appear enlarged as cells shrink but cling to each other.
The proposed solution is to remove the sodium from the extracellular fluid before the fixative arrives. Saline prewash fluid washes out the vascular space, but also the extracellular space, since saline moves readily between compartments. Sucrose at 10% by weight concentration is approximately isotonic, will not cause water to move into or out cells, and contains no sodium. A prewash with isotonic sucrose solution will wash out the extracellular sodium in all soft organs except brain. The blood brain barrier prevents sucrose from crossing out of the vascular compartment in brain. However, this can be overcome by increasing the pressure to 300 mm Hg, enough to open the blood brain barrier without changing the light or electron microscopic appearance of capillaries or blood vessels. And with the advantages of clearing the red blood cells within as little as 5-10 seconds, and allowing the switch to fixative earlier than at lower pressures. Fixative reaches the tissues at very little time delay after onset of anoxia.
Of course, isotonic sucrose very much changes the dynamic situation. Any ion or particle at higher concentration inside the cell than in the ion-free sucrose solution will begin to move out into the flowing fluid and be washed away. By the end of even a very short prewash, the cells are no longer “isotonic” on the inside. So to keep them the same size, the fixative should be in some concentration that would be hypotonic compared to normal cells, isotonic compared to the prewashed cells. In preliminary studies, whole organ expansion or shrinkage, depending solely on fixative tonicity after a pressure sucrose prewash, has been observed (Scouten and Cunningham, unpublished). For a more complete discussion of tonicity, see Scouten, O’Connor and Cunningham, 20063 or Scouten, 20094.
A fixative procedure that applies a controlled pressure higher than systolic blood pressure will yield better wash out of red blood, better perfusion with fixative, and a more uniform and consistent tissue quality between mammals of different ages, weights or previous histories than a procedure driven by gravity pressure or fixed flow rate.
- Cragg, B., Preservation of extracellular space during fixation of the brain for electronmicroscopy. Tissue and Cell 12(1): 63-72, 1980
- Medawar, P.B. The rate of penetration of fixatives. J R Microsc Soc. 61:46, 1941.
- Scouten, C.W. O’Connor, R. & Cunningham, M, Perfusion Fixation of Research Animals, Microscopy Today 14: 26-33, 2006
- Scouten, C.W. Histology on Research Animal Tissue.I In: A Practical Guide to Frozen Section Technique. Ed: Stephen R. Peters Springer Verlag, 2009.
- Short, C. E. Principles & Practice of Veterinary Anesthesia. Williams & Wilkins, Baltimore, 1987, page 456.
- Palay, S.L., McGee-Russell, S.M., Gordon, S. & Grillo, M, 1962, Fixation of Neural Tissues for Electron Microscopy by Perfusion with Solutions of Osmium Tetroxide. The Journal of Cell Biology 12: 385-410, 1962
- Van Harreveld, A. The extracellular space in the vertebrate central nervous system. In: The Structure and Function of Nervous Tissue (ed. G. H. Bourne) Vol 4, pp 447-511 Academic Press, New York, 1972