[Histonet] Perfusion debate

Wed Oct 8 13:11:33 CDT 2003

   Joining in the perfusion debate......... I have perfused mouse and 
rat CNS with a pump and had excellent results. I have never measured the 
pressure the pump puts out but I do use a syringe needle (into the left 
ventricle)  with a large diameter, 16 gauge for a rat, 20 gauge for a 
mouse. I think something close to the inside diameter of the aorta is 
required for good results. In my experience, high rate of flow is more 
important that pressure. I don't worry about washing out every last RBC, 
I don't think it can be done. I use a minimum amount of buffer (a few 
milleliters) as a prewash and I don't use vasodilators or 
anti-coagulents. No shrinkage problems for LM or EM. I have never tried 
sucrose as a prewash.
   I have also perfused rat kidney with just two jars high on a shelf 
above the sink. Beautiful EM's from that work, even from the renal 
medulla which has relatively low blood flow. Again, I did not measure 
the pressure but it was only about 90-100 mm Hg  (converting the height 
in inches to mm then converting from water to mercury using specific 
gravity).
   To summarize, use a high rate of flow and be sure the buffer (not the 
fix+buffer solution) you use is somwhat hypertonic. This last point is 
well documented in the literature.

Geoff

mucram11 <@t> earthlink.net wrote:

> Strange we did not have shrinkage problems with our set up.  Pam Marcum
>
> Original Message:
> -----------------
> From: Charles W. Scouten, Ph.D. cwscouten <@t> myneurolab.com
> Date: Wed, 8 Oct 2003 10:39:33 -0500
> To: mucram11 <@t> earthlink.net, frouwke <@t> sci.kun.nl,
> nancy.walker <@t> sanofi-synthelabo.com, histonet <@t> lists.utsouthwestern.edu
> Subject: RE: [Histonet] RE: shrinkage and Perfusion One system
>
>
> Yes, gravity will work, with 20% shrinkage of the brain, some shape
> distortion, and with a reddish tinge due to remaining red blood cells,
> which catalyze any HRP reaction, and autofluoresce.  OK for some 
> purposes,
> terrible for others, but not the optimum for any. 
> I have asked www.Histonet.org  to post a picture called Perfused Brains
> Annotated which shows whole rat brains, one fresh dissected out, one
> perfused by pressure sucrose and  4% paraformaldehyde/glut, and the 
> last by
> traditional gravity flow with saline prewash, and the same fixative.
>
> The last is reddish and shrunken compared to the middle brain. 
> Three ft of water translates directly, using the units converter at:
> http://www.sciencemadesimple.com/conversions.html
> to  67 mm Hg, below average diastolic pressure in mice or rats or 
> people. And some pressure is lost in the lines and needle.  Even in 
> living animals
> with cycles of systolic pressure, some capillaries occasionally get 
> plugged
> for a while and stop flowing.  That is part of the advantage of exercise,
> to keep the lines open.  It is extremely unlikely that completely open
> exposure to 67 mm Hg would in any way "blow" the vascular system of any
> mammal, or even clear out all the blood.  The blocked passages will stop
> fixative from reaching the surrounding tissue in that area, and lead to
> local deterioration in unpredictable and irreproduceible areas.
>
> The only advantage of sucrose over saline is to clear the extracellular
> fluid of sodium before fixative hits.  High pressure, 300 mm Hg, 
> applied to
> either saline or sucrose as the prewash, (there is never a reason for
> both), will thoroughly clear all red blood cells, but should not be 
> pumped
> up in advance the 'thump' the system, but increase over a few seconds to
> clear most blood before the blood brain barrier opens.  This prewash will
> take half the time of traditional prewash, and get fixative in sooner and
> more thoroughly.
>
> 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: mucram11 <@t> earthlink.net [mailto:mucram11 <@t> earthlink.net] Sent: 
> Wednesday, October 08, 2003 8:06 AM
> To: frouwke <@t> sci.kun.nl; Charles W. Scouten, Ph.D.;
> nancy.walker <@t> sanofi-synthelabo.com; histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] RE: shrinkage and Perfusion One system
>
> Hi,
> We never had a pump. We used gravity for over 2,000 rats so I think I can
> say it will work.  The height of the container or IV bottle used 
> seemed to
> be the key.  We controlled flow with the standard IV roller closure on 
> the
> tubing.  It was approximately 3ft above the area the animal was being
> perfused in, always under a hood or very well ventilated area. We looked
> for a steady drip in the tubing and out the cannula.  We did not open the
> valve all the way as it would blow out the vascular system. We did have a
> couple of students try wide open and prove the point very well. 
> The use of a saline solution with and without anticoag agents had been
> used.  If you are going to use sucrose I would use the pre-wash to get 
> the
> blood and red cells out.  Just to be sure you have all the small vessels
> cleared for the fixative or freezing technique. 
> Pam Marcum
>
> Original Message:
> -----------------
> From: Frouwke Kuijpers frouwke <@t> sci.kun.nl
> Date: Wed, 8 Oct 2003 12:47:14 +0200
> To: cwscouten <@t> myneurolab.com, Nancy.Walker <@t> sanofi-synthelabo.com,
> Histonet <@t> lists.utsouthwestern.edu
> Subject: Re: [Histonet] RE: shrinkage and Perfusion One system
>
>
> 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
>  
>

-- 
-- 
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 mcauliff <@t> umdnj.edu
**********************************************

-- 
--
**********************************************
Geoff McAuliffe, Ph.D.
Neuroscience and Cell Biology
Robert Wood Johnson Medical School
675 Hoes Lane, Piscataway, NJ 08854
voice: (732)-235-4583; fax: -4029 
mcauliff <@t> umdnj.edu
**********************************************