[Histonet] RE: shrinkage and Perfusion One system

Wed Oct 8 12:19:33 CDT 2003

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

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