Spinal cord dissection

[Thitus]

Hi,

I am trying to do IF on spinal cord cryosections and the first question is how one can dissect the (lumber) spinal cord without damaging it?

I have tried 'flushing out' the spinal cord from the isolated vertebral column using a needle attached to a 20ml syringe w/ PFA as well as cutting it out. The first approach never worked, the spinal cord never came out. As for the second method - I am worried that my cutting might cause a lot of damage.

My 2 questions are:

1. Is it necessary at all to remove the spinal cord from the bone and muscles or can I just section it within the surrounding tissue? Please note that the animals are perfused, which is why I think this shouldn't impair fixation.

2. If it IS necessary to isolate it - how do you do it??


Thanks a lot for your comments.
Thitus

[CanuckPhD]

For mice our lab has flushed out the spinal cord with the needle/syringe technique and the tissue was not too damaged. However, we found that this only works for fresh tissue, not perfused tissue.

For rats and perfused mice we remove the spinal cord from the body by cutting along its length, so that the vertebrae remain in a column. We strip off most of the tissue, to increase penetration of the PFA. After post fixation (24h) we perform a laminectomy (removal of the top of the spinal cord) to expose the spinal cord. We then gently remove it.

This method also has the advantage of allowing us to isolate specific areas of the spianl cord by counting the vertebrae. In this manner it is easier to compare specific areas as compared to flushing out the spinal cord.

Good luck

[Thitus]

Wow thanks, I greatly appreciate your advise! But there are still more questions:


>> After post fixation (24h) we perform a laminectomy (removal of the top of the spinal cord) to expose the spinal cord. <<

Which dissection instruments do you find most convenient for this step?

Do you remove the dura before doing the staining protocol?

Which landmarks of the vertebral column can I use to identify the lumber area of the spinal cord? At which vertebra does the spinal cord end in mice? Is there an atlas of the mouse spinal cord you could recommend?

I hope this is not asking for too much.

[CanuckPhD]

For dissection we use a couple of pairs of small scissors (with pointed ends) to snip the top of the vertebrae off and a pair of rat tooth forceps to hold it and apply a slight upwards pull on the spinous process. We use a blunt nose pair of scissors (Mayo) and large rat tooth forceps to remove the tissue and expose the spinal cord.

The dura is removed at the time of dissection as we process each spinal cord segment separately and embed the pieces in a standard manner. This allows us to compare the same areas between animals.

I don't the exact number but we usually take the last 6 vertebrae as the lumbar area. This may also capture some sacral segments as well but for our purposes it is sufficient.

Hope this helps. Good luck.

[ImmunoNYC]

Great advice given above!
 
Another probably less desirable option (but it still works for us non-neuroscience folks who dabble in it from time to time!) is to remove the whole vertebral column and fix whole (or perfuse depending on application). In this case we then decal using EDTA and section in whatever orientation we choose. Works nicely for my immunos and is especially nice when I also want to look at marrow and muscle simultaneously.

[kskristinesmith]

Step 1
Clean the vertebral laminae from approximately C7 to the sacrum. See your instructor if you need help performing the laminectomy. Open the dura mater to expose the spinal cord. Find a dorsal root ganglion, spinal nerve, and dorsal and ventral rami.

Step 2
Demonstrate the cauda equina.



Hope this helps.
kristine
fast slim

[hkennedy]

Hi there!

Does anyone have any references to articles describing spinal cord removal procedures (rats & mice) that you could point me toward?

Thanks!!