Hello Mathilde,

I worked mainly on gastrocnemius atrophy (a bit of tibialis atrophy).

The quadriceps is a big muscle, but I think the approach you describe makes sense. I focused on the middle part of "my" muscles, but analysing several sections from middle and either side (for example at one fifth of the length or so) like Mohamed described certainly is useful, but is more work.

My suggestion would be to induce "strong" atrophy (for example with Doxorubicin in IP or whatever you use), have a look where it is the strongest and then only keep that region for the next analyses.

Hi Mathilde,

we always focus on the mid-belly.

I don't have any experience working with murine models, but I can explain how we approach this issue in human research. Because we cannot remove an entire muscle and chop it up to study atrophy, imaging techniques provide non-invasive measures of measure atrophy on the level of the whole muscle. MRI for example can provide a volume measure of the whole muscle, whereas similar CT methods may carry too high a radiation dose, and are often limited to one or several transverse image slices. These slices often investigate the "mid-belly" or the location of the largest muscle girth, with the intent to capture the greatest number of parallel muscle fibres. Alternatively, muscle biopsies can be collected from willing participants for fibre-type staining however without collecting multiple samples on each participant, these biopsies may only be specific to only one muscle from the quadriceps muscle group.

Hi Mathilde,

Good question and indeed analysing the entire length of muscle Quadriceps will be tedious. I would go around the mid-belly region where individual muscle fibres oppose each other and analyse the entire cross-sectional area of myofibers of the sample replicates for analysis and further confirm the gene expression and/or the protein levels of atrophy markers.

Hope it helps.

Hi Mathilde,

it depends a bit on your exact research question. The fiber types can vary towards the distal and proximal sides of the muscle. This of course also depends on the exact transgenic conditions, age of the mouse etc.

To get an idea of how much this varies along the muscle, I would agree with Mohamed and quantify your measures in at least 3 or 4 slices (that are representative for the entire muscle). 

If it's not a problem, I would agree with the other answers, to take the mid-belly region in order to be able to quantify the largest number of fibers in one section.

It really depends on your question. You MUST begin with a control experiment to define fiber type and size variability along and across the muscle. Then look at the same thing among the quads. This gives you the variance values you will need to use the Power equations to calculate sample size--#slices #muscles #fibers--it takes time but is well worth it in the end. Your sample size might vary between wt and control. 

Hi Mathilde,

as suggested by Dr. Lieber, you surely need to perform a control experiment.

In terms of fiber type composition, in theory it makes sense to look at very different regions of the muscle. But I'm not sure that it really helps, when trying to study muscle atrophy, to average measurements of fiber type, size or number coming from very different regions of the muscle.

Moreover, in terms of fiber size, I don't think it is really a good idea to look too far from the mid-belly region. This because some/many fibers are no more parallel, when getting closer to the tendons. In transverse section, you can easily see this, because fibers tend to appear more elliptical. Measuring such kind of fibers, you're going to over-estimate the CSA.

I have a lot of experience with mouse tibialis anterior, gastrocnemius, soleus and rat soleus and EDL, but not with quadriceps. However, I usually I do something similar to what proposed by Mohamed, taking some sections starting from the mid-belly of the muscle, spaced by 1-200 micron (of course, take some serial sections, to be able to do all the kind of staining that you are planning).

One last thing, if you're planning to extensively measure CSA and fiber-type, there's a way to automate the procedure, using a freeware software called SMASH, as reported in the link down here. Alternatively, you can use ImageJ/Fiji, but things are a little bit more tricky.

Good luck!!!

http://www.ncbi.nlm.nih.gov/pubmed/25937889

I really want to thank ALL of you for your answers, they are helping me a lot! I wish you all the best in your own research,

Mathilde