I'm not familiar with craniotomies over the olfactory bulb. That said ... how do you perform the craniotomy currently: what type of drill bit, what drill pattern? Do you make a hole and widen it, or do you create a skull flap and remove that in one piece?

Use a dental drill and make a hole by circling the drill in such a way that you thin out the skull until u feel the skull is soft enough to be picked up by forceps.

as above, the use of a dental drill with gentle motion so that you can feel when nearly through gently pulling the drill up so as not to disturb any tissue below.

Thanks everyone for your suggestions!

I haven't been using a drill - currently I use a biopsy punch that makes a 3 mm hole that is exactly the size of the windows I am using. So I use the biopsy punch until I can feel that the skull is loose on all sides, but what I'm having trouble with is that even when the bone is no longer attached it often still seems stuck to some tissue underneath (and craniotomies over the olfactory bulb are particularly difficult I think because of the large blood vessels that run down the midline - if these are damaged at all, the animal often bleeds to death). I like using the biopsy punch because I think it gives me a lot of control, but do you think thinning the skull with a drill helps to disconnect the skull from any tissue underneath (maybe the vibrations from the drill help to kind of dislodge things)? 

A biopsy punch, interesting! I had not heard of this technique before, not sure how widespread it is, but I guess it is used for OB windows, as in: "Long-Term Imaging Reveals Dynamic Changes in the Neuronal Composition of the Glomerular Layer"

If you like, try this: using a dentral drill and a small drill bit (e.g. 0.5 mm if you can find that) drill the outline of your craniotomy (slightly larger than intended). Gradually trace the outline with the drill, making a channel in the bone. Gradually increase the depth all around the circumference, until it is very thin everywhere (thin enough to crack in a few places). Then cover with saline and gently use fine forceps to take hold of the "island" of remaining skull in the middle - you should be able to stick one tip into the skull flap from the side (into the softer part); alternatively, grab it however you deem fit. And now very gently lift the skull flap up and away; you can try twisting slightly. If it doesn't come off easily, stop, and check for spots where the channel isn't deep enough, and drill some more, then repeat the removal attempt.

I haven't tried that for OB, but it works well for other dorsal areas of the mouse skull. For a good window, I believe you want to avoid all bleeding during the craniotomy.

I don't know whether the vibration helps with separation, as you suggest ...

I might try the biopsy punch method and compare!

http://www.jneurosci.org/content/31/22/7967.full

I also hadn't heard of a biopsy punch until this forum! The dental drill is also very effective for thin skull preparations which provides optical access but leaves a very thin layer of skull at the very bottom to help protect the brain. Thin skulling is associated with much less inflammation/astroglioisis than a cranial window but it of course depends on your application. 

http://www.nature.com/nmeth/journal/v7/n12/abs/nmeth.1530.html 

Mark West's lab has created an open-source stereotaxic robot that could be very useful to you.

Jove: http://www.jove.com/video/51006/building-an-open-source-robotic-stereotaxic-instrument

Resources: http://www.eden.rutgers.edu/~mrcoffey/robot.html

I tried using the biopsy punch following Mizrahi's paper for olfactory bulb craniotomy over one or both bulb and I didn't have problems removing the flap bone. But actually In young animals (2-3 weeks old) I felt it was actually hard to stop at the right depth before damaging the dura and it often bled... So I went back to classical drill or reinforced thin skull which is much more simple. But it depends whether you want to access the bulb or just perform 2 photon recordings and if you want to keep the animals for chronic experiments...  

Hi Jenelle, 

Do you have a protocol for doing craniotomies with a biopsy punch? Our lab currently uses a drill to remove the skull over the cortex, but I think that a biopsy punch might be a much more efficient way to go!  

As far are your dura situation, I would suggest to be very careful that you are not going too deep. When I am drilling, I try to make the skull thin enough to give way under slight pressure. Ideally the skull will crack on its own without having to drill completely through. Perhaps using a biopsy punch for the initial shaving & then switching to a dental drill for the final delicate "cracking" stage would help to avoid dura damage. I also leave a slightly thicker area of bone intact to act as a hinge when I lift off the bone flap.

Anecdotally, I have found that when I lift the skull (especially near sagittal sinus), some vessels will "tear" causing some bleeding. I'm not sure how to avoid this.

Thanks for the biopsy idea!

cheers,

Valerie

I have also been performing craniectomy in mice, which is required in the Fluid Percussion Injury model. But the site of craniectomy in my work is between bregma and lambda, and between the sagittal suture and lateral ridge. I have been using a dental trephine of 2.5 mm and performing craniectomy manually. During trephination, we have to provide stability to the trephine for this purpose we have to use nylon chord (solid wire of nylon) of around 1.5 mm in diameter and of the same or little more in length, which has to be glued over craniectomy site using dermabond. This nylon chord helps us to easily pick up the bone flap without damage to the dura after gentle trephination. I think this method may help you...

Find the whole procedure in this article (Article Low-pressure fluid percussion minimally adds to the sham cra...

Hey everyone,

I've been doing craniotomies over V1 a lot and I use surgifoam absorbable gelatin sponge cut up into tiny little pieces and soaked in saline. It's helpful for soaking up any little bleeding that occurs. But mainly, I find it super helpful during the drilling process. I also irrigate a lot throughout drilling and once I've drilled to a point the skull is paper thin such that if I drill anymore I will hit the dura, I place some the saline-soaked sponge on the bone flap and leave it for 10-15 mins. When I lift, the bone flap is much easier to remove without any damage to the dura.

However, I've lately been experiencing some problems with mice with a genetic mutation. Their windows tend to get inflamed around 7-14 days post-op and then this inflammation clears up a bit between 14-21 days post-op. I am wondering if anyone has any tips and tricks for reducing inflammation? I use a dentsply sirona dental drill. I don't have much bone dust with this air drill so I don't think anything is contaminating the craniotomy or brain. I give them IP injections of dexamethasone and carprofen on the day of surgery, 24 hours later, 48 hours later, and a week after their second day of post-op injections. I'm trying to figure out different methods of improving the recovery of my windows. Any help would be appreciated!