Re: Method development for rat tibial bone.

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From:"Barry Rittman" <> (by way of histonet)
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happy thanksgiving. A few suggestions for a complex area.
As you mentioned crown rump length I am assuming that the measurements will
be on
developing rats rather that adults?  This may complicate some things due to the
open nature of intramembranous bone formation, the large number of cells
and the
rapid turnover of bone in developing rodents.

1.    Fixation. Buffered formalin seems to be a good overall fixative for bone.
2.    Decalcification I prefer EDTA pH 7.2 to 7.4 and for these specimens time
should not be too long.
3.    Staining.
General structure of epiphyseal plate progressive H and E to demonstrate growth
lines and reversion lines. For elegance trichrome especially Heidenhain's azan
with prior mordanting of sections in dichromate or Bouin's.
4.    Image analysis
Most image analysis programs will allow you to measure areas and perimeters of
Easiest method to demonstrate trabecular bone is a van Gieson without any
stain. For osteoid, I prefer a trichrome again without a nuclear stain.
Both these
simplify histomorphometric analysis especially if using a video image analysis
system. Nuclear staining while nice makes the image analysis that much more
The marrow cavities will vary in size and therefore need to decide on what
measurements you will need. Best while image analysis is being carried out to
measure size of marrow cavity or set a standard size frame to allow direct
comparisons between specimens. area of trabecular bone, thickness of
osteoid (as
will give an indication of rate of deposition). In adjacent section would
try to
measure numbers of osteoclasts and relate this to length of bone surface.
Numbers of  active and resting osteoblasts also indication of bone
formation. If
measuring only a limited length of bone surface need to standardize the
region as numbers can vary considerably.
Orientation of sections while cutting is critical as marrow cavity will vary in
appearance according to direction of cut. Often best to mark the outside of the
specimen with Indian ink to aid in standardization.
Measurement of calcified cartilage and associated calcified bone is often
the most
difficult measurement as the staining between the two is not as contrasty as we
would wish.

5.    Crown rump length easiest to measure by first photographing using a
setup with 2-3 photographs per specimen with calibration ruler and then measure
from photographs. here the important item is to standardize your measurements
especially if using animals of different ages.

Hope these comments help.
Barry wrote:

> Hi Histonetters-We have just begun working on methods development for the
> assessment of tibial bone parameters in rats and are wondering if there
>is any
> preferred methods out there.  Any particular decal solution being used to
> protect the integrity of the cell structures?  Any consistent way
>sections can
> be cut to examine the epiphyseal growth plate and other bone parameters what
> would allow a histomorphometric analysis?  Also, is anyone utilizing a
> crown-rump measurement and how do you do it?
> Many thanks in advance,
> Kate Rhodes
> SmithKline Beecham Pharmaceuticals
> 610-270-7340
> FAX 610-270-7202

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