Fresh                      Pressure Perfused                   Gravity Perfused

Perfusion pressure above physiological levels enables:

• Faster throughput-Faster fixation
• Complete washout of vascular compartment
• Automated or manual pressure control
• Sucrose or saline prewash
• Postwash if desired

Purpose

The NeuroFixer™ pressure perfusion apparatus used as described below optimizes fast, artifact free fixation of tissue for sectioning and microscopy.  The goal of the NeuroFixer™ is to preserve dynamic cells in as close to a snapshot of the position and shape of living cells as possible.  For  brain cells, the most artifact-free fixation is achieved by a high pressure (300 mm Hg) prewash with isotonic (9.5%) sucrose solution (Cragg, 1980), (Schwarzmaier, et. al.,2022) until the slight twitch ceases (very brief), then switch to paraformaldehyde (formaldehyde freely crosses cell membranes and is thus not a contributor to tonicity) in 9.5% sucrose until the tail is rigid to the tip. (Scouten, 2010). This procedure forces sucrose through the blood brain barrier (BBB),  necessary to wash out extracellular fluid, but thus damaging cells within the vascular tissues maintaining the BBB (Schwarzmaier,  et. al. 2022).  The NeuroFixer™, with phosphate buffered (PBS) saline instead of sucrose and at 150 mm Hg instead of 300 mm Hg, can be used for studies of diseases or drugs that may affect the BBB.  Sucrose should only be used at a pressure high enough to break the blood brain barrier, it will not cross the BBB to wash out the extracellular fluid unless high pressure above physiological is used.

Perfusion does not preserve cell internal metabolites, which requires high power microwave fixation for controlled fractions of seconds (Murphy, 2010).  

Gravity vs. peristaltic pump vs. controlled pressure perfusion.

   Gravity

The earliest and still common driver of perfusion fixation of research animal tissue was gravity.  This constant pressure depends on the height of the bottle fluid surface above the animal, minus pressure lost to resistance to flow in tubing and needle.  Commonly, but variably between labs, that would be 25-40 inches.  Ceiling height above a sink counter becomes an issue, and convenience of filling and replacing the bottles.  Blood pressures are commonly measured in units of mmHg.  Since 1 mmHg=.535776 inches of water,  40 inches of water would equal 40/.535776=75 mm Hg, minus pressure lost in tubing, needle, and animal.   75 mm Hg is about half of a high systolic blood pressure.  Frequent poor perfusions due to well below biological pressures, with incomplete red cell washout, as seen in this figure, has led many researchers to switch to peristaltic pumps, and use constant flow rate.

  Peristaltic pumps

Controlled flow rate pumps will generate whatever pressure is need to force that flow rate.  There are individual, age, and group differences in what flow rate would generate what pressure.   A successful flow rate for a group of animals must be developed empirically.  If a rat and a mouse, or a larger rat or one with some existing blockage, were perfused at the same flow rate, at least one would be a very poor perfusion.

   Controlled Pressure

In contrast, within limits, most mammals have similar blood pressures and similar upper and lower limits on short term tolerance for pressures. Any perfusion pressure that worked for a rat would work for a mouse.  Controlling the pressure, but at levels higher than gravity can be in a lab setting, given ceiling heights, is the better choice.  Why double high systolic pressure (300 mmHg)?  First, let’s talk prewash fluid.

Prewash Fluid, Saline vs Sucrose?

Plasma without solids is mostly physiological saline.  So saline is the natural choice for prewash, right? Maybe natural, but usually not the right choice.   Sodium continuously leaks into cells, but is actively pumped out by energy requiring molecular pumps to achieve something like a 10:1 exterior: interior ratio of sodium ions.  Balanced tonicity is maintained by other ions and particles in the cell.  At onset of prewash, without red blood, cells become anoxic. Consequently, the pumps shut off.  Sodium is on unlimited supply.  Running saline for 2 minutes has sodium running into cells and not coming out.  Water follows into cells to balance tonicity.  Cells inflate to maximum size.  All extracellular spaces are drained of fluid, and cells expand into them (Cragg, 1980).  Then crosslinking fixative starts, binding proteins that are adjacent, presumably including those in adjacent cell walls.  At offset of perfusion pressure, some fluid will drain out of the relaxing organ and cells, and cells bound together will shrink some.  No extracellular space detectable by electron microscopy remains or reopens in the brain (Cragg, 1980). The result is organ shrinkage and distortion of normal morphology.  Look at the third ventricle in any fixed brain section compared to those in the atlas of Paxinos, which are unfixed to avoid distortion and maintain stereotaxic coordinates.

Suppose we could immediately replace saline in the vascular compartment and the extracellular space with a fluid that was isotonic, contained no sodium, and could not leak into cells?  Thus, little swelling, even after onset of anoxia.  Ordinary table sugar, sucrose, cannot cross cell membranes and has no sodium ions.  It is isotonic at a concentration of 9.5% in distilled water.  Non-ionic, no buffering needed. There is a catch.  Sucrose at blood pressure levels can’t cross the blood brain barrier (BBB), and so can’t replace the sodium fluids in the extracellular fluids compartment.  However, if the pressure is pushed just high enough to force sucrose fluid through the BBB, say 300 mm Hg (Cragg, 1980) it can wash out all extracellular fluids.  At this pressure, all blood is very rapidly removed through the vascular compartment and the extracellular fluid is replaced.  Seconds, not 2 minutes.  Fixative can then reach the cells sooner to stop autolysis. Under an ordinary microscope, tissue sections look excellent.  Morphology compares to Paxinos Atlas’s (prepared with fresh unfixed tissue).

Schwarzmaier et. al., 2022 tested several levels of perfusion pressure, using standard solutions of PBS prewash and paraformaldehyde fixative, and 2 minutes of prewash.  They compared several types of artifacts found in perfused brain tissue with confocal microscopy and sheet light microscopy.  Pressures below physiological levels, comparable to gravity perfusion, were uniformly worse results then pressure above those levels.   These authors recommend pressures around 150 mmHg. Although most artifacts were lowest at highest pressures, the artifacts below were worse at 300 mmHg pressures:

1)            Incidents of lengths of vasospasm, blood vessels constricted and blocking flow over a short area. This was with saline prewash for 2 minutes. Could the blood vessel muscle have constricted if there was no sodium in the vicinity?  Remember, a sudden inflow of sodium triggers excitable cells to react.

2)            Cells lining blood vessel walls and creating the blood brain barrier showed significant damage.  Yes, that had to be done to release sucrose from the vasculature.  High pressure perfusion is not suitable for studies of damaged, diseased, drug or any other effects on the  blood brain barrier.  The NeuroFixer™ can be used at other pressures with saline prewash.

Citations:

Cragg, B.,  Preservation of extracellular space during fixation of the brain for electron microscopy.  Tissue and Cell 12(1): 63-72, 1980

Murphy, E J. Brain fixation for analysis of brain lipid-mediators of signal transduction and brain eicosanoids requires head-focused microwave irradiation: and historical perspective. Prostaglandins & other Lipid Mediators 91 (2010) 63–67

Paxinos and Watson.  The Rat Brain in Stereotaxic Coordinates, 7^th Edition,  2013

Paxinos and Franklin’s the Mouse Brain in Stereotaxic Coordinates

Scouten, C., O’Connor, R., Cunningham, M.  Perfusion Fixation of Research Animals.  Microscopy Today volume 14, 2006.

Scouten, C.W. (2010). Frozen Section Technique in the Animal Research Setting. In: Peters, S. (eds) A Practical Guide to Frozen Section Technique. Springer, New York, NY.

Susanne M. Schwarzmaier, Maximilian R.O. Knarr, Senbin Hu, Ali Ertürk, Farida Hellal, Nikolaus Plesnila.  Perfusion pressure determines vascular integrity and histomorphological quality following perfusion fixation of the brain. Journal of Neuroscience Methods, Volume 372, 2022

AlHowaish, AlSudai, Khounganian, & AlMuraiki.  Histological Evaluation of Restylane Lyft Used as a Scaffold for Dental Pulp Regeneration in Non-Infected Immature Teeth in Dogs.  Materials 15(12):4095, 2022.