[Histonet] better perfusion and inclusion---thanks to y'all


Just to say thanks to numerous contacts that made helpful suggestions and
give feed back.

*Thanks Gail Callis for her suggestions to reduce inclusion schedules .
This did in fact reduce dilation of mouse brain ventricles.

*Thanks to helpful suggestions of Charles Scouten, Geoff McAuliffe and
Christophe Guerin, we greatly improved our perfusion technique with mouse
brain resulting in better histology : less blood vessel collapse, cytoplasm
retraction visual particularly around oligodendrocytes but also neurons,
and better integrety of hippocampal cell layers.

What worked for us?
We set up a perfusion system based on physiological blood pressure which
will take into account size variation between young and older animals. It's
simple and cheap, too! Just supply pressure with the rubber bulb and
manometer ( we used  a medical blood pressure unit, without the cuff!)
remember to use stiff tubing and adjust the catheter to just fit into the
aorta of the size of your animal. Don't worry you don't have to pump much
to keep up the pressure, especially with mice (a little more with rats).
This system resulted in better evacuation of red blood vessels than
perfusing at 2.5 ml/min (110mm Hg in our 35g mice resulted in a speed of
about 1.3 ml/min).

Trials to break the blood-brain barrier with brief treatments of sacharose
or mannitol with high pressure did nothing to improve the histology rather
made it worse. What did give more reproducible results in terms of
histology  was extending the perfusion time from 10 to 20 minutes. I put up
a picture in the Histonet web gallery (http://pathcuri1.swmed.edu).

So in conclusion systems like Perfusion One that allow you to perfuse on a
pressure basis do better flush out red blood cells, breaking the brain
blood barrier with various treatments didn't help but maybe we didn't do it
right.  Longer perfusion times really reduce variability.

thanks again,

Nancy Walker
Molecular Pharmacology

Sanofi-Synthelbo Research
B.P. 37 Labége Innopole

tel : (33)561004179  fax :(33)561004001

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