[Histonet] Murine brain perfusion

[scoop <@t> mail.nih.gov]

Dear Histonetters,

I do mouse perfusion using a 24 gauge tubing adaptor (no sharp ends) 
in the left ventricle and cut the right atrium open (you need 
somewhere for the perfusion fluid to exit the sytem).  But I have 
always wanted to try cannulating the aorta  or vena cava - what size 
catheter or tubing do people use for that?  Also, is it possible to 
cannulate the aorta through the left ventricle?

Thanks,
Sharon

>It has been years, but here goes.  We used a 2%
>paraform+1% glut mixture in a 0.1M buffer (a
>Sorensen's phosphate was our choice) at about pH 7.2.
>The setup was fairly simple:  a drip bottle was filled
>with warm (actually RT) fixative and elevated to about
>6ft.  A second bottle held saline for doing a flush
>and the 2 bottles were connected via a simple 2-way
>valve.  A 26ga needle was inserted into the ascending
>aorta and the abdominal aorta was cut to allow
>outflow.  The flush is run until clear (probably no
>more than 30-60sec) at which point the valve is
>switched to the fixative.  Fixation is adequate after
>about 5min (max).  The brain is removed and placed in
>fresh fixative for at least 1hr.  (This technique
>should actually give whole body fixation.)  Coronal
>sections were taken of the brain and either processed
>for electron microscopy, into paraffin for routine
>H&E, placed on a vibratome and 50um sections obtained
>for tracer localization (evaluating blood-brain
>barrier integrity), etc.  Probably the diciest part of
>the technique is inserting the cannula correctly into
>the ascending aorta and clamping it in place--we used
>small vascular clamps for this. 
>
>Another life, many years ago.
>
>Roger Moretz, Ph.D.
>Dept. of Toxicology
>Boehringer Ingelheim Pharmaceuticals, Inc.
>Ridgefield, CT
>
>--- Jackie M O'Connor <Jackie.O'Connor <@t> abbott.com>
>wrote:
>
>>  How do you do it?   We would like to perfuse whole
>>  mouse brains with
>>  fixative (what kind?) in order to make 2mm coronal
>>  sections to visually
>>  examine the brain for  therapeutic changes as soon
>>  after harvest as
>>  possible.    Would someone be willing to walk me
>>  through their protocol? 
>>  I don't have any experience with this at all - and
>>  they're looking to me
>>  for answers.
>>  Happy Friday - Jackie
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