[Histonet] Perfusion debate

[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,
>
Yes.

> 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. 
>myNeuroLab.com 
>5918 Evergreen Blvd. 
>St. Louis, MO 63134 
>Ph: 314 522 0300  
>FAX  314 522 0377 
>cwscouten <@t> myneurolab.com 
>www.myneurolab.com 
>  
>

Gotta get ready for class.

Geoff

>
>-----Original Message-----
>From: Geoff McAuliffe [mailto:mcauliff <@t> umdnj.edu] 
>Sent: Wednesday, October 08, 2003 1:12 PM
>To: histonet <@t> pathology.swmed.edu
>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 
>gravity).
>   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
>
>mucram11 <@t> earthlink.net wrote:
>
>  
>
>>Strange we did not have shrinkage problems with our set up.  Pam Marcum
>>
>>Original Message:
>>-----------------
>>From: Charles W. Scouten, Ph.D. cwscouten <@t> myneurolab.com
>>Date: Wed, 8 Oct 2003 10:39:33 -0500
>>To: mucram11 <@t> earthlink.net, frouwke <@t> sci.kun.nl,
>>nancy.walker <@t> sanofi-synthelabo.com, histonet <@t> lists.utsouthwestern.edu
>>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 
>>purposes,
>>terrible for others, but not the optimum for any. 
>>I have asked www.Histonet.org  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:
>>http://www.sciencemadesimple.com/conversions.html
>>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 
>>plugged
>>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 
>>pumped
>>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. myNeuroLab.com 5918 Evergreen Blvd. St. 
>>Louis, MO 63134 Ph: 314 522 0300  FAX  314 522 0377 
>>cwscouten <@t> myneurolab.com www.myneurolab.com
>>
>>-----Original Message-----
>>From: mucram11 <@t> earthlink.net [mailto:mucram11 <@t> earthlink.net] Sent: 
>>Wednesday, October 08, 2003 8:06 AM
>>To: frouwke <@t> sci.kun.nl; Charles W. Scouten, Ph.D.;
>>nancy.walker <@t> sanofi-synthelabo.com; histonet <@t> lists.utsouthwestern.edu
>>Subject: Re: [Histonet] RE: shrinkage and Perfusion One system
>>
>>Hi,
>>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 
>>the
>>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 
>>the
>>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 frouwke <@t> sci.kun.nl
>>Date: Wed, 8 Oct 2003 12:47:14 +0200
>>To: cwscouten <@t> myneurolab.com, Nancy.Walker <@t> sanofi-synthelabo.com,
>>Histonet <@t> lists.utsouthwestern.edu
>>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 
>>red
>>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 
>>mm300Hg....
>>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 
>>sucrose?
>>
>>Frouwke Kuijpers
>>
>>----- Original Message ----- From: "Charles W. Scouten, Ph.D." 
>><cwscouten <@t> myneurolab.com>
>>To: <Nancy.Walker <@t> sanofi-synthelabo.com>;
>><Histonet <@t> lists.utsouthwestern.edu>
>>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 
>>with
>>the sucrose.  Time is of the essence, tissue deterioration begins
>>immediately upon anoxia.  Switch to fixative when the animal's muscles 
>>cease
>>to move.
>>
>>Charles W. Scouten, Ph.D.
>>myNeuroLab.com
>>5918 Evergreen Blvd.
>>St. Louis, MO 63134
>>Ph: 314 522 0300
>>FAX 314 522 0377
>>cwscouten <@t> myneurolab.com
>>www.myneurolab.com
>>
>>
>>-----Original Message-----
>>From: Nancy.Walker <@t> sanofi-synthelabo.com
>>[mailto:Nancy.Walker <@t> sanofi-synthelabo.com]
>>Sent: Friday, October 03, 2003 8:12 AM
>>To: Charles W. Scouten, Ph.D.; Histonet <@t> lists.utsouthwestern.edu
>>Subject: shrinkage and Perfusion One system
>>
>>
>>Hello,
>>
>>    
>>
>>>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 
>>mmHg
>>to break the blood brain barrier and wash the extracellular fluid (and 
>>red
>>blood cells). Switch shortly thereafter to fixative. The perfusion takes
>>the same time as the traditional procedure, and accomplishes the 
>>favorable
>>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,
>>
>>Nancy
>> 
>>
>>    
>>
>
>  
>

-- 
--
**********************************************
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 
mcauliff <@t> umdnj.edu
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