[Histonet] RE: shrinkage and Perfusion One system

[Frouwke Kuijpers]

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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