[Histonet] RE: Histonet Digest, mouse pup euthanasia with CO2

From:"Jones, Lynne"

Hello to the list -
I have little histology experience, but I've dealt with complications arising from both anesthesia and euthanasia in lab animals.  Standard protocols don't work for every model.

Neonatal rodents are very resistant to hypoxia (death can take up to 50 minutes, depending on age and strain).  Our institutional policy is that neonatal rats and mice must remain inside the CO2 chamber for a minimum of 20 minutes after the gas flow is turned off if CO2 is the sole agent used for euthanasia.  Many Institutional Animal Care and Use Committees suggest consideration of alternative methods for neonates, or that CO2 anesthesia be followed by a physical method.

FWIW, the paper below describes some of the biological effects associated with different euthanasia techniques.

Based on the description of the brains, my suspicion is that the pups sat too long post-mortem before being fixed, or (going way out on a limb) that acidosis might have created problems if the formalin wasn't buffered.

Lynne Jones

-----Original Message-----
From: histonet-bounces@lists.utsouthwestern.edu [mailto:histonet-bounces@lists.utsouthwestern.edu] On Behalf Of histonet-request@lists.utsouthwestern.edu
Sent: Monday, February 11, 2008 12:07 PM
To: histonet@lists.utsouthwestern.edu
Subject: Histonet Digest, Vol 51, Issue 17

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Today's Topics:

   1. Fwd: [Histonet] Re: Mouse pups and processing (Greg Dobbin)
   2. Verhoeff/ Trichome procedure (karenadams@comcast.net)
   3. RE: Verhoeff/ Trichome procedure (Fail, Mildred M.)
   4. Iron staining (Till, Renee)
   5. RE: OT: the weather (Ingles Claire)
   6. prostate biopsy problem (Joyce Cline)


Message: 1
Date: Mon, 11 Feb 2008 08:54:11 -0400
From: "Greg Dobbin" 
Subject: Fwd: [Histonet] Re: Mouse pups and processing
Content-Type: text/plain; charset=US-ASCII

Greg Dobbin, R.T.
Chief Technologist, Histology Lab
Dept. of Laboratory Medicine,
Queen Elizabeth Hospital,
P.O. Box 6600
Charlottetown, PE    C1A 8T5
Phone: (902) 894-2337
Fax: (902) 894-2385

There is some merit in doing the right thing rather badly,
but absolutely none in doing the wrong thing excellently!

>>> Greg Dobbin 2/11/2008 8:53 AM >>>
Hi Pam,
I participated on a project about 10 years ago where the rat pups were
anesthetized using an overdose of halothane followed by exanguinationn
(carotid and/or renal arteries). Here are the 2 relevant references:
Comp Med. 2001 Apr;51(2):134-7.
Histol Histopathol. 2002 Oct;17(4):1067-76.
Good luck.

Greg Dobbin, R.T.
Chief Technologist, Histology Lab
Dept. of Laboratory Medicine,
Queen Elizabeth Hospital,
P.O. Box 6600
Charlottetown, PE    C1A 8T5
Phone: (902) 894-2337
Fax: (902) 894-2385

There is some merit in doing the right thing rather badly,
but absolutely none in doing the wrong thing excellently!

>>>  2/10/2008 9:57 AM >>>
Thank you Amos and I can appreciate your problem.  Your discription of
prefect.  I have also referred to the outer skin as lacy in appearance
attempting to section it.  The other point I would make is the brain is
with no form as if it is autolysized totally.

I have tried splitting the abdomen and it does not help or is so
minimal it
doesn't help much.  I have tried changing the processor and always
other tissues at the same time to show the PI it is not the reagents
paraffin.  We have processed other pups before this over the last year
and know
they were not killed with CO2.  They work great and section great (I
the abdomen).

I am at a loss and finally turned to all of you on HistoNet help.  You
and even though we may not have the answer yet we will I am sure of it.
I know
this is an ongoing problem for researchers with pups.  Maybe with this
thing being a popular way for sacrificing animals we have hit on
something that
happens in pups and not adults.  I had not really ever worried about it
now as I did my own sacrifice and it was fine.  I wish they weren't so
small I
would attempt prefusion with a 2cc syringe and blunt needle as a last

I want to THANK everyone who has and will take the time to answer and
help me
with this issue.  At least I can show the PI our lab is not the only
one with
the problem and we are all attempting to find a way out of it.  I think
all I have seen we should be more aware of how the pups are sacrificed
and note
any differences between the way our PI's sacrifice them.  Maybe all of
material submitted to the Histology area for research on pups and other
should be recorded and we might see differences they don't in method.
Recommendations from IACOCC do not take our area of histology into
consideration as they are controlling the most humane and painless
method of
sacrificing animals over all.  Perhaps one of us can get a paper out of
this or
a talk in the future that helps everyone.

Who knows I even checked the phase of the moon this last time and it
did not

Thanks again and still looking.  If I solve this you will all be teh
first to


Quoting Amos Brooks :

> Pam,
>      We were having a similar problem with one of our researcher's P0
> I don't know how they sacrificed them, but they too dod not process
at all.
> They were squishy!
>      I reccommended they either bisect or at least make a good
> incision prior to fixation. It still isn't perfect (is it ever?), but
> seems to be a bit better. This allows better solution penetration.
Give it a
> whirl and let us know how it goes.
> Amos
> Message: 13
> Date: Fri, 08 Feb 2008 15:52:34 -0500
> From: Pamela Marcum 
> Subject: [Histonet] Mouse pups and processing
> To: Histonet@lists.utsouthwestern.edu
> Message-ID: <>
> Content-Type: text/plain; charset="us-ascii"; format=flowed
> I have been struggling with this for a while and need some help.  We
> currently have a project with 0 day mouse pups that are allowed to
> born normally and then sacrificed with CO2.  We had several groups
> earlier that were sacrificed a different way and they processed and
> sectioned beautifully.
> These don't seem to fix well, dehydrate, clear or infiltrate worth a
> darn.  Since this is my first time using CO2 for sacrifice I need to
> find out if it causes a problem or if I am just losing it.  I have
> not ever had this problem before and even re-processing does not
> help.  I know they are not infiltrating as they are floating in
> paraffin at the end if I remove them from the processor to a vat of
> paraffin.  They will not sink.  The pups are 1.2 to 1.3 grams each.
> If you can suggest a better way to sacrifice them please let me
> know.  Killing the mother and perfusing her is not an option as
> are not our mice.  They are being given as favor so I am limited to
> some extent.
> This was an overnight process with slightly altered alcohols to 45
> minutes each, 1 xylene at 30 minutes and 2 Shandon Xylene Substitues
> at 10 hour each to 4 paraffins at 45 X2 and 1 hour X2.
> Best Regards,
> Pamela A Marcum
> Manager, Histology Special Procedures
> University of Pennsylvania
> School of Veterinary Medicine
> R.S. Reynolds Jr.  CORL
> New Bolton Center
> 382 West Street Road
> Kennett Square, PA 19348
> Phone - 610-925-6278
> Fax     - 610-925-8120
> E-mail - pmarcum@vet.upenn.edu

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