Re: [Histonet] Perfusion debate

From:Geoff McAuliffe

Continuing ..........

Charles W. Scouten, Ph.D. wrote:
The large diameter needle into the ventricle sounds great. 
Works for me.
High flow rate sounds good to get clean of red blood cells, but how high is high, and in what species and body size?
Good question, I never did any real calculations. Let's see, blood volume is about 8% of body weight in man so I will stick with that number unless someone has a better (documented) one.  I will also assume that cardiac output is proportional in man/mouse/rat. Man is 5 liters/minute and the circulating blood volume is about 5 liters. For a 30 gram mouse (30x8%), about 2.4 ml of blood circulating every minute.When I perfuse a mouse I pump in about 60-75 ml in 5 minutes, lots more than the circulating volume over the same amount of time. For a 300 gram rat (about 24 ml of circulating blood) I pump in a minimum of 300 ml in 5 minutes, again much more than would circulate over the same amount of time. I rarely have a perfusion "fail" and I don't have problems with shrinkage, distorted cells, tissues, etc. I suspect that the high volumes I perfuse in a short amount of time are the cause. I have never measured the pressure my pump puts out with the needle sizes I use, perhapsI should find a gauge of some sort.
Of course, pressure and flow rate are closely related,
 the third determining variable is cardiovascular resistance, which is related to size, gender, species, health condition, etc.  A mouse would have far more resistance to a given flow than would a pig. 
I don't think so. One can have very high flow and very low pressure. A large diameter hose will move a lot of fluid  with very low pressure. Cardiovascular dynamcs (see your table below)  are reasonably similar in mammals, given good health. Gender differences would be small, but measurable. Resistance is primarliy determined by the (cumlative) cross-sectional area of the vessels,  hence the pressure drop in the arterioles just before to the capillary beds. Any fluid, not just blood, will behave this way. It's Poiseuill's Law.
 However, as might be guessed, all mammals have very close to the same average blood pressure.  See the table below, (pointed out to me by histonetters, thank you again):
The following is a table of average blood pressure for several species (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 what would happen if you perfused a pig with the same flow rate that works well on a mouse, or vice versa.  But you can use the same pressure to perfuse a pig or mouse, and get comparable results. 

 Controlling pressure is safer and more predictable.

A high flow rate means high pressure, 
No! Related yes, but not that way. To get one liter of solution through a 16 gauge needle in one minute will require a lot less pressure than moving the same amount of fluid through a 25 gauge needle in the same amount of time.
but the operational definition of high flow rate must depend on the species and body size, among other variables. High but safe pressure is 200 mm Hg in all of the above.  The blood brain barrier is reliable broken in hamsters and rats at 300 mm Hg.  What do you bet for the rest?
At 300 mm Hg, I come very close to washing out every last RBC in Hamster or Rat brain.  HRP shows maybe one in each whole brain coronal 60 micron thick section.  There is no noticeable (by me) damage to capillaries, but maybe a better anatomist could see it.

You do have shrinkage in the CNS, it is well documented. 
References, please.
So when I remove the skull from an animal perfused my way why isn't the brain 20% smaller than a fresh brain?
 You don't see it as a problem because it has always been there, and you haven't worked with fixed sections without it.  See Konig and Klippel for "inevitable shrinkage", and ask Paxinos why his atlases are done only on fresh, not fixed tissue.  But you can have both fixed and no shrinkage.  You EM sections showed little or no extracellular space.  
My EM sections show an amount of extracellular space appropriate for the tissue. Not much in CNS, lots in the dermis.
Actually, the 5% sucrose is slightly hypertonic, isotonic is something like 4.94 by weight.  I had never heard that point, though, can you tell me more about the documentation on that? 
Arborgh, et al, J. Ultrastructure Research 56:339-350, 1976. Not perfusion fixation, but verifying that the osmolality of the buffer the fixative is in, not the osmolality of  fixative, is important. There is lots of work on fixation and shrinkage, time in fixative, pH of fixative, etc. done in the 1950's and 1960's. Both model systems and tissues were used.
 Kidney is different, there is no blood brain barrier, and near isotonic sucrose would replace the extracellular fluid without the high pressure. 
The blood-brain barrier is not a barrier to pressure, it is much more of a diffusion/metabolic barrier.
 Isn't the active sodium pump limited to only exciteable cells? 
No, all cells have an ATP-driven sodium-potassium pump to keep Na outside and K inside.
 If so, this formalin shrinkage thing would only be relevant for neural or muscle cells.  Does anybody know if kidney or liver, for example, shrink with perfusion?

 Charles W.  Scouten, Ph.D. 
5918 Evergreen Blvd. 
St. Louis, MO 63134 
Ph: 314 522 0300  
FAX  314 522 0377 

Gotta get ready for class.


-----Original Message-----
From: Geoff McAuliffe [] 
Sent: Wednesday, October 08, 2003 1:12 PM
Subject: Re: [Histonet] Perfusion debate

   Joining in the perfusion debate......... I have perfused mouse and 
rat CNS with a pump and had excellent results. I have never measured the 
pressure the pump puts out but I do use a syringe needle (into the left 
ventricle)  with a large diameter, 16 gauge for a rat, 20 gauge for a 
mouse. I think something close to the inside diameter of the aorta is 
required for good results. In my experience, high rate of flow is more 
important that pressure. I don't worry about washing out every last RBC, 
I don't think it can be done. I use a minimum amount of buffer (a few 
milleliters) as a prewash and I don't use vasodilators or 
anti-coagulents. No shrinkage problems for LM or EM. I have never tried 
sucrose as a prewash.
   I have also perfused rat kidney with just two jars high on a shelf 
above the sink. Beautiful EM's from that work, even from the renal 
medulla which has relatively low blood flow. Again, I did not measure 
the pressure but it was only about 90-100 mm Hg  (converting the height 
in inches to mm then converting from water to mercury using specific 
   To summarize, use a high rate of flow and be sure the buffer (not the 
fix+buffer solution) you use is somwhat hypertonic. This last point is 
well documented in the literature.

Geoff wrote:

Strange we did not have shrinkage problems with our set up.  Pam Marcum

Original Message:
From: Charles W. Scouten, Ph.D.
Date: Wed, 8 Oct 2003 10:39:33 -0500
Subject: RE: [Histonet] RE: shrinkage and Perfusion One system

Yes, gravity will work, with 20% shrinkage of the brain, some shape
distortion, and with a reddish tinge due to remaining red blood cells,
which catalyze any HRP reaction, and autofluoresce.  OK for some 
terrible for others, but not the optimum for any. 
I have asked  to post a picture called Perfused Brains
Annotated which shows whole rat brains, one fresh dissected out, one
perfused by pressure sucrose and  4% paraformaldehyde/glut, and the 
last by
traditional gravity flow with saline prewash, and the same fixative.

The last is reddish and shrunken compared to the middle brain. 
Three ft of water translates directly, using the units converter at:
to  67 mm Hg, below average diastolic pressure in mice or rats or 
people. And some pressure is lost in the lines and needle.  Even in 
living animals
with cycles of systolic pressure, some capillaries occasionally get 
for a while and stop flowing.  That is part of the advantage of exercise,
to keep the lines open.  It is extremely unlikely that completely open
exposure to 67 mm Hg would in any way "blow" the vascular system of any
mammal, or even clear out all the blood.  The blocked passages will stop
fixative from reaching the surrounding tissue in that area, and lead to
local deterioration in unpredictable and irreproduceible areas.

The only advantage of sucrose over saline is to clear the extracellular
fluid of sodium before fixative hits.  High pressure, 300 mm Hg, 
applied to
either saline or sucrose as the prewash, (there is never a reason for
both), will thoroughly clear all red blood cells, but should not be 
up in advance the 'thump' the system, but increase over a few seconds to
clear most blood before the blood brain barrier opens.  This prewash will
take half the time of traditional prewash, and get fixative in sooner and
more thoroughly.

Charles W.  Scouten, Ph.D. 5918 Evergreen Blvd. St. 
Louis, MO 63134 Ph: 314 522 0300  FAX  314 522 0377

-----Original Message-----
From: [] Sent: 
Wednesday, October 08, 2003 8:06 AM
To:; Charles W. Scouten, Ph.D.;;
Subject: Re: [Histonet] RE: shrinkage and Perfusion One system

We never had a pump. We used gravity for over 2,000 rats so I think I can
say it will work.  The height of the container or IV bottle used 
seemed to
be the key.  We controlled flow with the standard IV roller closure on 
tubing.  It was approximately 3ft above the area the animal was being
perfused in, always under a hood or very well ventilated area. We looked
for a steady drip in the tubing and out the cannula.  We did not open the
valve all the way as it would blow out the vascular system. We did have a
couple of students try wide open and prove the point very well. 
The use of a saline solution with and without anticoag agents had been
used.  If you are going to use sucrose I would use the pre-wash to get 
blood and red cells out.  Just to be sure you have all the small vessels
cleared for the fixative or freezing technique. 
Pam Marcum

Original Message:
From: Frouwke Kuijpers
Date: Wed, 8 Oct 2003 12:47:14 +0200
Subject: Re: [Histonet] RE: shrinkage and Perfusion One system

Start the prewash at low pressure, and pump up to 300 mmHg
to break the blood brain barrier and wash the extracellular fluid (and 
blood cells).

Charles: next question: we don't do the perfusion by a pump, just by
gravity. You said the pressure for the 10% sucrose must go till 
Well we dont' have a pump: what will your advice be than?
Start with the saline solution to get rid of the blood and than the 

Frouwke Kuijpers

----- Original Message ----- From: "Charles W. Scouten, Ph.D." 
To: <>;
Sent: Monday, October 06, 2003 3:53 PM
Subject: [Histonet] RE: shrinkage and Perfusion One system

The saline prewash would be just lost time.  It accomplishes nothing that
will not be accomplished by the sucrose, which is equally effective at
washing out the blood (more effective given the high pressure). Start 
the sucrose.  Time is of the essence, tissue deterioration begins
immediately upon anoxia.  Switch to fixative when the animal's muscles 
to move.

Charles W. Scouten, Ph.D.
5918 Evergreen Blvd.
St. Louis, MO 63134
Ph: 314 522 0300
FAX 314 522 0377

-----Original Message-----
Sent: Friday, October 03, 2003 8:12 AM
To: Charles W. Scouten, Ph.D.;
Subject: shrinkage and Perfusion One system


From the website mentioned in C. Scouten's mail I recuperated this info:
The shrinkage occurs when a prewash with physiological saline is followed
by fixative. The first action of fixative on the cell membrane is to shut
off the sodium pump proteins. Sodium rushes into the cell, followed by
water to maintain tonicity, and cells swell and expand. Membrane proteins
are fixed and crosslinked to neighboring cells in the swollen position.
Later, the cells stabilize and contract, and pull other cells with them.
The result is gross shrinkage, local distortion and torn membranes with
lost cellular contents from some cells.

Tissue Shrinkage can be completely prevented if the extracellular fluid
with ions is replaced by a nonionic isotonic solution that cannot 
enter the
cells. This can be accomplished by prewash with 5% (isotonic) sucrose in
distilled water. Start the prewash at low pressure, and pump up to 300 
to break the blood brain barrier and wash the extracellular fluid (and 
blood cells). Switch shortly thereafter to fixative. The perfusion takes
the same time as the traditional procedure, and accomplishes the 
results bulleted above.

So if I'd like to try out the perfusion techniques that are made 
simple by
the Perfusion one system, what would you suggest:   first do a
physiological saline solution, followed by a prewash with 5% sucrose and
then PFA, or go directly to 5% sucrose then PFA?

thanks for your advice,




Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029

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