mouse spinal cord removal revisited

From:Gayle Callis

Just wanted to share a successful protocol.  A few weeks ago, I asked how
people were removing a whole mouse spinal cord from vertebral column.  Lots
of appreciated answers with the majority not using the method described
here.  We are 100% successful with the method most people did not advise
using.  The method we used is published, and uses a syringe/needle to push
spinal cord out from thinnest area of cord towards the thick, wider
cervical area.   

Dull an 18 guage hypodermic needle (used a fingernail file that has 4
grinding grits) to remove sharp ends and bevels.  Leave a bit of bevel on
needle but use fine grit to smooth off cutting edges.  Do not cut needle
end off squarely.  The narrowed, dull bevel helps enter the tiny opening in

Fill 20 ml syringe/dull needle combo with PBS as 10 mls didn't work as

Disarticulate vertebral column just below skull and at hip level;  remove
ribs and viscera. Rinse blood away with PBS in order to see the cord.  Use
scalpel to cut across vertebra, clean cuts are manadatory across cord,
shattered bone makes smooth entry into lumen more difficult without a snug

The spinal column target area just anterior (forward of) to hind leg area
OR just posterior to lumbar region of column.  The lumbar region is thicker
but that is not apparent unless bone is carefully cleaned of muscle, etc
(this was not necessary). This is where the needle is inserted.  

The needle must fit snugly into lumen of vertebra, cord should be visible
unless bloody (rinse with PBS!). 
Do not force needle too much or bone splits - tight fit is important.  Push
on plunger (this took some major pushing force while holding slippery
tissue) and inject PBS into column.  The spinal cord slipped out cleanly
and perfectly whole.  The cord was NOT damaged.  Be sure to use a large
container to catch PBS/cord, messy when cord slides out very fast due to
pressure behind it, PBS tends to everywhere  - don't lose cord down a sink.

This method has eliminated the need for longer NBF fixation and expensive,
very time consuming decalcification of boney vertebral column. 

The only problem I can see with this method is the need to have a complete
cord all the way to tail. One might have to use a smaller diameter needle
closer to openings near tail (caudal vertebrae) or go back to long
fixation/decalcification method. 


Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
S. 19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367 (lab with voice mail)
406 994-4303 (FAX)


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