John Kiernan jkiernan <@t> uwo.ca
Sat Oct 15 23:21:47 CDT 2005

I like your comments, Charles. I too read the paper by
Eichenbaum et al in BioTechniques 39(4): 487-500 (2005)
and was surprised that a slow 1 ml intracardiac injection
of Somogyi & Takagi's (1982) buffered
cocktail would fix a mouse's brain well enough for EM. 

Eichenbaum et al describe the technique in great detail, and
emphasize that deviations and errors will result in failure.

Anecdotally: perfused glutaraldehyde-containing fixatives
don't clog blood vessels in the way that formaldehyde-only
solutions sometimes do. Several hands-on academic researchers
that I knew in bygone days made this assertion. I've never 
had trouble perfusing any water-based fixative except for
bad placement of the needle or cannula, and that happens
more often with mice than rats, because of the smaller size.

Unduly high perfusion pressure can certainly cause artifacts.
I've seen (by EM) huge pericapillary spaces in brain tissue
following perfusion done with a syringe. The thumb pressure
waa unknown but probably was far to high. Perfusion from a 
bottle 1 metre above the bench does not induce this artifact.
A metre of water is a crude approximation to the pressure
in a large artery of a living rodent.

Eichenbaum et al injected their fixative through a 27-gauge
needle. That's much thinner than the ones used for 
conventional perfusion, when the emphasis is on getting a
substantial flow rate. The thin needle and the long, narrow
recommended syringe would make it easier to do the injection 
slowly. Glutaraldehyde reacts rapidly with proteins, and
probably gets rid of the blood-brain barrier in seconds.

Geoff McAuliffe is amazed at the small volume of the 
injected fixative. One ml has to be 20 to 30 percent 
of the blood volume of a 30 gm mouse. 

John Kiernan
Anatomy, UWO
London, Canada

Charles Scouten wrote:
>  I downloaded and read this article.  I am also surprised to read the
> authors statements that it works so well.  I would have some questions
> and concerns remaining after reading the article.
> The fixative arrives while the red bloods cells are still present.
> Don't they crosslink to proteins in the blood vessel linings, and
> adhere?  Does this procedure really wash out red blood cells more
> cleanly than the traditional procedure? With no prewash?  Sufficient to
> run a clean HRP reaction with red cells catalyzing noise reactions?
> Where do they go?  Since no escape vessel is cut, the excess fluid must
> accumulate somewhere.  There is a clear experience and skill requirement
> here to press the plunger at the right force and rate.  There is a need
> for an instrument to control the expulsion if this works as well as the
> authors seem to be saying.  I would like to hear from them about this.
>  The authors miss-attribute to Cragg the contention that pressure causes
> swelling.  I can assure them from experience that this is not the case,
> and will publish on that and other issues shortly .  The traditional
> procedure does indeed produce swelling and distortion, followed by
> shrinkage, due to fixation of the sodium pump proteins and the inrush of
> sodium ions.  Cragg used a very high pressure, 300 mm Hg, to force
> isotonic sucrose across the blood brain barrier to get rid of sodium in
> the extracellular fluid before the fixative arrived, and that avoided
> the swelling and subsequent shrinkage.  I note that the pressures used
> by the authors in pushing on the syringe plunger is unknown, but great
> enough to force fluid in against physiological blood pressure.
> I was impressed that the ventricles in the samples shown were not
> swollen, much like unfixed, unperfused tissue.  How do we know the blood
> was out?  H&E staining works fine in blood filled tissue, and in unfixed
> tissue.  Does GFAP? I guess the EM shows that at least cortex as fixed
> and preserved. Why is postimmersion fixation unnecesary in this tissue?
> Both perfusion methods get fixative everywhere fast, but traditional
> gets progressively better fixations with days in post perfusion
> fixative.  That wouldn't happen with this procedure?
> If you do try this, I would very much like to hear the outcome.
> Cordially,
> Charles W.  Scouten, Ph.D.
> myNeuroLab.com
> 5918 Evergreen Blvd.
> St. Louis, MO 63134
> Ph: 314 522 0300 x 342
> FAX  314 522 0377
> cwscouten <@t> myneurolab.com
> http://www.myneurolab.com
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Geoff
> McAuliffe
> Sent: Thursday, October 13, 2005 1:27 PM
> To: Favara, Cynthia (NIH/NIAID); Histonet
> Subject: Re: [Histonet] MOUSE BRAIN FIXATION
> Years ago my colleagues who worked on mouse bloood would withdraw up to
> 1 ml of blood from the mouse heart in a similar manner, but hitting the
> left ventricle does take some practice.
> I am a little amazed at the low volume of fixative as well, but tilting
> the animal so that the head is lower than the heart would help.
> Note that the resolution of the photos is not all that good.
> Geoff
> Favara, Cynthia (NIH/NIAID) wrote:
> >I have this paper and hope to try this next week. Note that the
> >fixative is picric acid/paraformaldehyde/gluteraldehyde. I would love
> >to get great fixation with 1ml and without opening the chest cavity. I
> will let you know!
> >
> >c
> >
> >Cynthia Favara
> >903 South 4th Street
> >Hamilton, MT 59840
> >406-363-9317
> >
> >-----Original Message-----
> >From: Adam Perry [mailto:kaleid11 <@t> yahoo.com]
> >Sent: Thursday, October 13, 2005 8:20 AM
> >To: histonet <@t> lists.utsouthwestern.edu
> >Subject: [Histonet] MOUSE BRAIN FIXATION
> >
> >I downloaded the technical report on this variant perfusion technique.
> >I'm amazed that you can get good fixation of the brain with 1 ml of
> >fixative pumped through the heart without clamping off descending
> >aorta, etc.  Has anyone tried this technique (or similar variants), and
> >what kind of results were obtained?
> >
> >I'm planning on trying it in a week or so, because I would love to get
> >good brain fixation with so little time, fixative and waste
> >production...but am wary...
> >
> >Adam Perry
> >
> >Department of Physiology and Biophysics University of Illinois Chicago,
> >IL 60612
> >
> >There have been several inquiries regarding fixation of mouse brains
> >recently.
> >
> >A short technical report has just been published in BioTechniques:
> >
> >Minimally invasive method for murine brain fixation Eichenbaum KD et al
> >Biotechniques 2005; 39(4): 487-500
> >
> >After simple registration, it can be accessed at
> >http://www.biotechniques.com <http://www.biotechniques.com>
> >
> >Ronnie Houston
> >Director of Anatomic Pathology
> >Bon Secours HealthPartners Laboratories
> >5801 Bremo Road
> >Richmond, VA 23226
> >(804) 2877972
> >(804) 2877906 - fax
> >ronnie_houston <@t> bshsi.com
> >
> >
> >
> >
> >---------------------------------
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> --
> --
> **********************************************
> Geoff McAuliffe, Ph.D.
> Neuroscience and Cell Biology
> Robert Wood Johnson Medical School
> 675 Hoes Lane, Piscataway, NJ 08854
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