About Perfusion

8/4/2019 About Perfusion

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Pressure Perfusion Application Note

In order to make histological observations of tissue morphology, the tissue must be fixed, sectioned, mounted on

microscope slides (or processed for electron microscopy) and stained to enhance visibility of all or selected cells. Without

fixation, tissue constituents begin to breakdown almost immediately upon onset of anoxia; i.e., decrease of oxygenformerly provided by circulating blood. Rapid fixation of tissue as soon after the loss of blood supply as possible

minimizes tissue changes initiated by anoxia.

Perfusion is a way to take advantage of the cardiovascular fast channel to every cell in the body to facilitate rapid fixation

in a way not possible with immersion of biopsy tissue in fixative (Cammermeyer, 1960, Garman, 1990). Biopsy or

autopsy tissue samples must be submerged in a fixative bath and left for a period of time. Fixatives (the most common

being a solution of formaldehyde) penetrates tissue at a rate of about 18 mm in 25 hours (diffusion = 3.6 * time)(Medawar, 1941; Baker, 1958).

After penetration, progressive formaldehyde crosslinking of proteins proceeds over about 25 hours, and fixative binding

and crosslinking may continue asymptotically for up to 7 days (Fox, 1985; Helander, 1994). Thus, the reproducibility of

results under the microscope will depend on the handling time prior to immersion in fixative, the thickness of a tissue

block, the density and type of tissue, and on the temperature of the fixative. Note also that immersion fixed tissue is less

cross-linked in the center of the block than on its surfaces. Overexposure to fixative can weaken immunolabeling and

HRP (horseradish peroxidase, a protein commonly used to label cells) activity. Underexposure to fixative renders thetissue friable and difficult to handle for histologic procedures; moreover, many stains are incompatible with underfixed

tissue (e.g. HRP reaction) .

Red Blood Cells

Red blood cells remaining in tissue hinder fixative penetration and obscure a clear view of the cells unique to the tissue

being studied. They also react similarly to target cells in many histologic procedures. For example, red blood cells

catalyze peroxidase reactions and stain along with HRP containing cells. Red blood cells therefore contribute to

background staining in such reactions (Mesulam, 1978). Red blood cells are also notorious for autofluorescence, and

luminescence along with fluorescent labels. Thus, two common methods used by researchers to label specific cells alsolabel remaining red blood cells. In addition, blood remaining in frozen fixed tissue softens it, and makes sectioning more

difficult and variable. It is thus very desirable to wash out all red blood cells.

Fixation by Perfusion

Tissue from animals sacrificed for research purposes usually has had blood cells removed, and fixative infused, by

perfusion. Animal researchers commonly perfuse the tissue transcardially by infusing isotonic salineinto the heart of a

deeply anesthetized animal, and cutting blood vessels returning to the heart. This washes the blood out of the circulatory

system. They then take advantage of the open vascular channel to perfuse with fixative. This fast exposure to fixative,

distributed evenly throughout the animal, allows experiments to be more reproducible and the tissue to be easier to stain

evenly throughout. Note, however, that human biopsy or autopsy tissue can not be pre-perfused. Rather, the tissue must

be immersed in solution, resulting in uneven fixation that is difficult to section and may not stain reliably (Garman RH,1990).

Despite the long-standing practice, and the advantages, of perfusion in animal research, it is not a perfected procedure.

Usually, variable amounts of red blood cells remain in brain and other tissues. Flow of perfusate is commonly driven bygravity or peristaltic pump. Amount or pressure of fluid flow is commonly not controlled or noted. Variance could cause

fixative to be unevenly distributed in the tissue. Thus, quality of perfusion may depend on differences in the perfusion

apparatus and procedure. Perfusion with formaldehyde solutions by traditional methods causes an elimination of

extracellular space, for reasons described below, and corresponding shrinkage of tissue.


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Control of Pressure vs. Flow Rate for Perfusion

Gravity flow or a peristaltic pump pressure are the common controlled means of applying pressure to the perfusion fluid.

Gravity provides a constant pressure, while a peristaltic pump provides a constant flow rate regardless of resistance. The

flow rate and pressure can not both be controlled, of course. The flow rate equals the pressure divided by the

cardiovascular resistance. Cardiovascular resistance is highly variable between and within species, while blood pressure

is comparatively very consistent (Short, 1987, Green, 1979). Therefore, it is easier to select a pressure that is appropriateacross species and individuals than it is to select a flow rate. The following is a table of average blood pressure for severalspecies (systolic/diastolic) (Green, 1979).

mice 113/81 rats 116/90 hamster 150/110

rabbit 110/80 dog 112/56 cat 120/75 baboon 148/100 rhesus 160/127 pig 170/108.

Consider the extreme case of a flow rate used in rats being applied in a pig. Pressure would be minimal. The prewash

fluid would find a few channels through, and trickle out, but blood washout would be very poor. Consequently, fixative

would not flow into many blocked capillaries, and autolysis would be in progress before fixative reached the tissue by

diffusion. Clearly, the flow rate selected must take into account the cardiovascular resistance of the animal, and beadjusted to generate a reasonable pressure.

Now consider applying 200 mm Hg fixed pressure in either species. This is well above average blood pressure in both

species, but in a range that can occur naturally without immediate damage. The resulting flow rate will be dramaticallygreater in the pig than in the rat, but washout and fixative distribution will be excellent in both cases. Capillaries are

opened and blood pushed out by pressure, not flow rate. Fixative only enters opened capillaries.

Cardiovascular resistance will vary widely between species, genders, strains, and individuals within strains. It wil

depend on previous exercise, body weight, fat percentage, and other variables. A fixed perfusion flow rate in the

physiological range will thus result in systematic bias, by gender, weight, or any variable influencing cardiovascula

resistance, in the quality of perfusion achieved. How much should the flow rate be adjusted to compensate for

cardiovascular resistance differences for a rat 100 grams heavier than another rat?

An inexpensive flow meter could be incorporated in a pressure controlled apparatus to allow control and/or reading of

flow rate. If the pressure and flow rate are recorded, the cardiovascular resistance could be calculated. This may be

relevant research data when the animal has been exercised or given other treatments that may affect cardiovascular

resistance. We have measured the average flow rate through a 250 gram rat with 300 mm Hg applied is 120 ml/min.

Traditional Gravity Perfusion Apparatus

Although the most commonly used apparatus for perfusion is simple (gravity-driven fluid flow from two bottles is merged

into one flow by a tubing Y connector, and regulated by clamps), it is surprising that no commercial apparatus is offered

for whole animal perfusion, given the ubiquity of the procedure in animal research. No manual offers directions for

reservoir height, tubing size, or needle gauge. Every lab procures parts and assembles their own apparatus, with differingideas of what may be important. Commonly, two containers with tubing attached to the bottom are set on a shelf at an

arbitrary height. Both bottles are connected to the upper arms of a Y connector by tubing. Tubing clamps between the

bottles and the Y connector enable control of which fluid is flowing. Tubing from the lower arm of the Y connector is

connected to a plastic syringe barrel from which the flange has been removed. A gavage needle is installed on the other

end of the syringe barrel.

The Math


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The pressure of perfusate entering the animals vasculature is determined by gravity and bottle height, minus pressure lost

to the tubing and the needles resistance to flow. Commonly, but variably, the bottles are 25-40 inches above the work

area on which the animal lies. Gravity pressure on an optimistic 40 inches of water translates to 76 mm Hg, a relatively

low constant pressure for the mammalian vascular system. Some pressure is lost in flow through the tubing and more

through the needle, which may offer as much resistance to flow as the open vascular system (which would mean the

animal might get as little as half the gravity pressure, e.g., 38 mm Hg pressure drop across the vascular system). This

may account for the inefficiency in removing red blood cells from brain capillaries. The prevailing system does notprovide enough pressure to wash out the blood. A pressure of 200 mmHg would require that the bottles be 9 ft. above

the animal, not possible in most lab spaces. Many researchers use a peristaltic pump to drive the fluids, rather than

gravity. Variable pump speeds and thus flow rates and pressures are used and are not standardized. Flow rate is

controlled rather than pressure. Optimal flow rate will depend on species, age, gender, cardiovascular condition, and any

other factor that can affect cardiovascular resistance.

Given the importance of perfusion as the starting point of almost all of animal histology, the nuisance of red blood cells,

and poor tissue working quality following poor perfusion, it gets surprisingly little attention in the methods section of

published papers. It is a factor in the quality and intensity of labeling for HRP reactions, fluorescence reactions, and any

immuno reaction. Pressures and heights, tubing diameters, cannula gauge, and flow rates vary among labs, but commonlyare not reported.

Shrinkage

On the electron microscopic level, brain cells and processes perfused with standard methods as described above appear in

apposition with each other, with very little extracellular space. Several lines of evidence including resistance studies and

cell counts against living volume show that the living brain is about 20% extracellular space (Van Harreveld, 1972). In

perfused and fixed brain tissue, using traditional protocols, this space is absent, and the brain is reduced in size by about

20%. This was and is accepted by most scientists as an unavoidable consequence of tissue processing, and is described as

such in the stereotaxic atlases This method needs some comment. It inevitably implies shrinkage caused by embeddingand staining. Shrinkage can not be equalized by enlargement because, for physical reasons, the extent of shrinkagediffers in the various constituents of the brain (Knig and Klipple, 1967). Later sections in Knig and Klipple made itclear the formaldehyde was the part of embedding and staining that caused the shrinkage. As a result, Knig and

Klipple, 1967 could not provide accurate stereotaxic coordinates that can be applied to living brain.

Paxinos and Watson (1998) avoided this problem by working only with fresh frozen tissue, and not fixing. Of course,

many histological reactions do not work with fresh tissue. Proteins will be coagulated by the mounting process, but for

purposes of gross anatomy, this works fine. The widest distance across any coronal section of whole brain is about 13

mm in Knig and Klipple, while this distance is 16 mm in Paxinos and Watson (1998). Although there are age and

weight differences, the brain size of adult rats does not change significantly with advancing age (Paxinos G, Watson C,

Pennisi M, Topple A, 1985) and cannot account for this discrepancy. Rather, the 20% difference may be attributed to

differences in tissue preparation for the Knig and Klippel (1967) atlas vs. the Paxinos and Watson (1998) atlas. The

perception that fixation induced shrinkage is inevitable is widely held to this day, and is the prevailing wisdom

(http://www.mbl.org/atlas247/atlas247_start.html). In fact, it is not inevitable, it can be avoided,and should be.

Preserving the Extracellular Space, Avoiding Shrinkage and Distortion

Cragg (1980) described a perfusion procedure that avoids the shrinkage and loss of extracellular space, but required a

more complicated apparatus. Perhaps because the articles title portrayed it as a technique for electron microscopy, and

perhaps because it required putting together specialized apparatus not available as a commercial unit, this technique has

not come into common usage for routine animal histology.


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Most, if not all, cell types maintain an active process of pumping sodium out and potassium in. The cytoplasm is highly

sodium depleted compared to the extracellular fluid. This gradient is mediated in an energy using process by a large (120

kilodalton ) protein protruding through the cell membrane. Cragg reasoned that one of the first effects of an aldehyde

fixative arriving into the extracellular fluid would be to denature the exposed cell surface membrane proteins mediating

the cellular sodium pump.

As a result of sodium pump cessation, sodium rushes into the cell down the osmotic gradient. Water also comes in tobalance tonicity. This in turn results in swelling of cells and cellular processes. Because cellular volume increases,

extracellular volume decreases and cells abut each other. Proteins in membranes are cross linked between cells. Later,

when membranes are fully permeable and equilibrium is restored, the cells return to their original size, and pull

neighboring cells in with them. The result is the observed 20% whole organ shrinkage.

Cragg proposed that if the extracellular fluid could be replaced by a non-ionic (or at least low sodium) isotonic fluid that

could not cross cell membranes before the arrival of the fixative, the in-rush of sodium and water caused by exposure to

aldehydes would not occur. Common sucrose at 9.25% concentration by weight meets this criterion, being isotonic with

body fluids but without ions.

Unfortunately, any fluid, including sucrose, which cannot cross cell membranes also cannot cross the blood-brain barrier

during perfusion, and thus cannot replace the extracellular fluid in brain. However, the blood brain barrier may be brokenby high pressure, without rupturing blood vessels. Cragg describes a procedure employing a peristaltic pump, a pressure

gauge, and manual regulation of the flow rate in order to deliver a pressure of 300 mm Hg. The prewash solution is

isotonic sucrose instead of saline. The pressure is increased after the start of flow to reach 300 mm Hg, which renders the

brain blood vessels permeable to sucrose. A washout of blood followed by extracellular fluid, which is replaced by

isotonic sucrose, follows. A lowered pressure can be used to deliver standard fixative solution of

formaldehyde/glutaraldehyde in neutral buffered solution, delivered at ~100 mm Hg. This perfusion procedure resulted inbrain tissue sections in which the 20% extracellular space was preserved. The same procedure and solutions, with the

exception that the pressure during the prewash with sucrose remained at or below 100 mm Hg, resulted in the usual loss of

the extracellular space. However, Cragg did not describe the effect of either procedure on the gross morphology of the

brain, on overall shrinkage, on red blood cell retention, or histological analysis on the light microscopic level.

Tissue Other than Brain

The sodium pump is a property of cells generally. Thus, the mechanism described above to result in shrinkage and

distortion as a result of traditional fixation applies to all tissues with a few modifications. Brain has about 20%

extracellular space. Other tissues likely have less, (e.g. skin) and will shrink correspondingly less. The brain is more

variable in consistency, with differing densities of gray and white matter throughout. Thus, it will likely distort more as

result of shrinkage than other more homogeneous organs. In brain, unlike most other tissues, localization is usually a

much more important issue in histology than cell morphology, and distortion a more serious problem. Red blood cells

react with or interfere with specific cell stains (HRP, immunoflurescence) very important to the neuroscientist; the high

pressure of the Perfusion One apparatus is very effective at clearing red blood cells. In organs without the blood-brainbarrier, plasma fluids and extracellular fluids mix more readily at physiological pressures. It would thus not be necessary

to pump the pressure up to 300 mmHg, or even above physiological range, to replace the sodium ions with sucrose in

organs other than the brain. It is thus fair to say that this particular perfusion apparatus and protocol is of significantlymore value to the neuroscientist than to those studying other organs.

The Perfusion One still has advantages for scientists working outside the brain. In setting up a lab, it is efficient to buy

what you need rather than design, source parts, and build it. For effective fixation of organs, the red blood cells must be

removed in order to let fixative penetrate throughout the vascular system; the high pressure available with this system is

very effective at clearing the red blood cells to allow homogeneous fixation. Fixative must arrive as soon after anoxia

begins as possible, to avoid deterioration; clearing the blood fast allows the fixative to begin to flow sooner, and with less

obstruction.


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C: Perfusion One Development

In the late 1980s, this author experimented with the Cragg protocol with a goal of getting more red blood cells out of the

brain for cleaner HRP reactions (Scouten & Malsbury, unpublished). Lacking a peristaltic pump, one-liter bottles of

sucrose at 4 C and fixative at room temperature were pressurized with a handheld rubber bulb, as used in blood pressure

measurement apparatus, regulating pressure directly by an air hand pump, rather than by flow rate. A mercury manometer

was used to monitor pressure level. Three-way valves and clamps were used to enable switching fluids. Bottles wereseparate, and connected by tubing.

Hamsters were anesthetized deeply and then perfused by inserting a large gavage needle through the heart into the

ascending aorta, and clamping it into place. Pressure was pumped up to 300 mm Hg, over about 5 or 6 seconds (it is not

desirable to pre-pressurize; since the blood should be evacuated before breaking the blood brain barrier). The animals

were perfused with about 500 ml of sucrose, after which flow was switched to fixative and the perfusion continued with

about 500 ml of fixative at 100 mm Hg.

The results were dramatically noticeable in several ways. Upon removal of the brains, their gross appearance was much

larger and whiter than we were used to. Previously, brains perfused at low pressures with a saline prewash, and fixed by

paraformaldehyde/glutaraldehyde fixative, had a shrunken, reddish look, and a harder consistency. With the new

procedure, coronal sections of hamster brain were larger and more anatomically correct. Ventricles were slits rather thanswollen balloons. Sections would no longer fit side by side on 1 inch width slides, but had to be arranged lengthwise on

the slide. HRP reactions on this tissue had very low background, and stained cells were strongly reactive. See Figure 2

and Figure 3.


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Labeled fiber tract in cross section

Negligible background, no red blood cells,

Labeled axons crossing the midline below

the optic chiasm

Dense cells

HRP reaction product in cut axons or

terminals

Figure 2. Low power darkfield photomicrograph of brain section from a hamster perfused with

pressurized sucrose, showing HRP labeled cells and fibers. Total magnification 14x. Note the near

absence of red blood cells and negligible background staining.

Red blood cell in capillary

Dark background, devoid of stain

Dense, strongly labeled large cells

HRP reaction product within cut axons or

terminals

Figure 3. High power darkfield photomicrograph of HRP labeled cells and fibers in hamster brain

perfused with pressurized sucrose. Total magnification 200x. Note the vivid staining and dark

background.


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We have recently designed and released the Perfusion One, a simple-to-use device to facilitate the pressure sucrose

perfusion procedure. Dr. Miles Cunningham used it to perfuse the brains shown in the attached pictures, Figures 4 and 5.

Note the center brain in Figure 4 is whiter, freer of red blood cells. Examine the cerebellum and rostral cerebrum to see

the shrinkage in the gravity perfused brain on the right. Figure 5 shows two sections from a comparable level in two

different brains, the left one perfused with gravity, the right one with the pressure perfusion technique. The swollen

ventricle is an index of shrinkage, note the thickness of cortex, and the vertical distortion. The pressure sucrose perfused

brain on the right is the more preserved morphologically.

Fresh Brain Pressure Perfusion Gravity Perfusion Gravity Perfusion Pressure Perfusion

Figure 4. Perfusion by different methods Figure 5. See Paxinos Plate 45 for Fresh

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