[Histonet] Perfusion techniques in mice/rats

Wed Oct 1 09:33:51 CDT 2003

What tissue are you after?  For brain, I used a modified version of the
EM protocol published by Cragg, 1980.  He found a way to avoid shrinkage
due to formalin fixation.  

When the fixative arrives, the sodium pump proteins on the outside of
the cell are the first to be fixed.  Sodium rushes into the cell down
the concentration gradient, and water rushes in to maintain tonicity.
The cell swells 30%, and fixation occurs with crosslinking to proteins
in neighboring cells.  Later, when equilibrium is restored and the cells
shrink back, they drag their neighbors back with them.  The result is
20% gross shrinkage of the brain, and loss of extracellular space.  

Cragg reasoned that if you could replace the extracellular fluid with
isotonic fluid without sodium, the shrinkage would not occur at all.
Isotonic sucrose (close to 5%) fills the bill, but will not replace
extracellular fluid because of the blood brain barrier.  This can be
overcome if the sucrose is pumped through at 300 mm Hg to overcome the
blood brain barrier without breaking blood vessels.  

We offer a suitable apparatus to implement this plan, see the link
below, and a protocol.  It has the salutary effect of completely and
thoroughly washing out the red blood cells.  You use sucrose for the
prewash instead of saline, very briefly because of the high pressure and
flow rate, then get the fixative to the cells more rapidly than
traditional methods.  

Gravity flow is too weak, 150 mm Hg translates to 6.7 ft of water.  In
your lab space, can the water reservoir be 6.7 ft above the animal on
the sink, without raising the ceiling?  Or to implement the Cragg
perfusion, you would need 13.4 ft. from animal to sucrose.   Peristaltic
pumps control flow rate, which does not correct for vascular resistance
of the animal, and the correct flow rate is not obvious.  Try the
Perfusion One:

http://www.myneurolab.com/myneurolab/mnl_products_detail.asp?idproduct=4
71001&catdesc=Histology+Equipment&CatThreeID=674&CatOneID=4&subcatdesc=S
acrifice+Equipment&idsubcategory=21

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 <mailto:cwscouten <@t> myneruolab.com>  
www.myneurolab.com 

-----Original Message-----
From: Noel D. Clark [mailto:Noel.Clark <@t> ORTHO.UAB.EDU] 
Sent: Wednesday, October 01, 2003 7:45 AM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Perfusion techniques in mice/rats

Was hoping to get input on the various perfusion techniques used by
other research labs for rodents.  As always, any help is greatly
appreciated.  See you in Louisville!

Thanks in advance,
Noel

Noel Clark, M.A., HTL (ASCP)
1919 7th Ave South
Orthopaedic Research Laboratory
Center for Metabolic Bone Disease
University of Alabama at Birmingham
Birmingham, Alabama 35294

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