Hi, I am currently learning to do electrophysiological recording on mice visual cortex, and sometimes the cortex bleeds and swells after the craniotomy and removal of the meninges. I want to know if bleeding is the major cause of the swelling cortex, and how to prevent the cortex from swelling.
Dear Zhou,
It is important not to cause any damage to the brain tissue-paranchyma at any stage of surgery. First remove the overlaying skin, then the bone carefully with care not too cause damage to the underlying membranes or excessive bleeding that may increse the pressure. Before cutting duramater, make sure all the bleeding is stopped- if the bleeding is coming from the bone, use wax to block it. A cautorizer with a small tip and surgucell may help with soft tissue bleeding. Once you remove the duramater, you will get to the piamater with many fine vessels and capillaries, make sure you do not touch or pinch this membrane. From now on you will have to keep the brain tissue moist either by warmed up saline or inert oil such as silicon oil. Another point is to use/make electrodes with long and thin shafts, so that the tissue damage due to multiple electrode penetrations will be limited-otherwise large diameter electrodes will make a large hole-damage on the surface, especially if you need to go deep into the brain.
Please have a look at my earlier studies and thesis for details of steretaxic surgery and methodology.
Best wishes.
Refik
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I endorse Refik's answer. Additionally, pay attention to the anesthetic management of the animal, e.g. avoid hyperventilation.
I have to say, I do not know about mice, as I work with rats. And here I do disagree with the presented answers. I do see swelling with and without intubation... I have the feeling it is the exposure time of the brain. The whole idea of an intracranial implantation is to go past the meninges, ergo damage is part of the game. We do see swelling with big craniotomies, but not anymore with only 0,8mmø skull holes, which are useful and big enough when you know where to go. No mapping with these size holes... The dura is removed inside the hole by a bent syringe needle tip (make it L-shaped according to the skull thickness). Good luck!
Dear Zhou,
once you have identified coordinates that work for you, then do exactly what Ulrich wrote! Make the hole as small as possible and you will have zero problems.
The main problem is bleeding. The second problem is long term exposure of an unprotected brain surface slowly drying out.
Large craniotomies can be done with zero bleeding, even when removing the entire skull surface. And the dura can be lifted using tiny eye scissors and then cut and folded away to one side.
As you can imagine, this takes lots and lots of practice!
But If you really want to go that way, then an excellent way to practice before doing any real surgery on living animals is removing the eggshell with a drill bit from raw eggs without piercing the underlying membrane. Drilling into e.g. chicken bones is another good exercise. Once you have become good at drilling, you can progress to dead animals. It also helps watching someone who can already do large craniotomies well.
Unfortunately, the best "trick" to prevent brain swelling is to prevent any bleeding in the first place. For that you have to perform a perfect craniotomy and make zero mistakes. And to be able to do that you need to practice a lot. Using a surgical stereo microscope is highly recommended!
Also, while drilling, the skull surface needs to be cooled using room temperature PBS or physiological saline solution. And after you have done a perfect craniotomy you have to constantly keep the brain surface moist using 37°C warm PBS or physiological saline solution.
Dear Zhou,
I only do tract tracing, so my experience is with very small craniotomies--just large enough for a 33 ga needle. If you don't have anyone handy to show you how to do large craniotomies in mice, you can check the videos on JoVE if you have access. There were some good ones (I don't currently have the links) for long term in vivo imaging with the glass coverslip implants that show how to make a fairly large hole. They also suggest which drill bits to try for the craniotomy. This is important since mouse skulls are a lot thinner than rat skulls.
Good Luck!
Jill
Dear Zhou,
following my initial post, this video shows the described procedure performed on a rat.
https://www.youtube.com/watch?v=655jRVPD3Qg
For doing it that well on a mouse you need even more practice.
Good Luck!
Olaf
Dear Zhou,
I was working with the technique Ulrich and Olaf described during my PhD thesis on 68 rats. It is really a matter of flushing the surgical area with physiological buffer. When you look from the tissues´ perspective, a swelling occurs by influx of liquid provoked by free substances derived from tissue damage. As larger the damage as bigger the effect. Multiple factors contribute to this scenario and you can really dismantle it, by reducing the concentration of swelling-causing agents due to flushing with physiological buffer. Not liters every minute but cover the surgical area in continuous intervals (several minutes, appropriate to your protocol and the temperature conditions on the ROI --> evaporation) with the buffer. Soak away supernatant (I have had a big drop of buffer in the surgical field, that disables optical check by refraction) with sterile sweps but keep it glossy and prevent by doing so any drying of the tissue. Drying will lead to even more swelling, due to tissue signals, that shall keep the wound wet when it dries. By doing so, you will have automatically a clearance of swelling-provoking agents. It must be done continuously during surgery, as the tissue tries to reach hemostasis all the time.
Hope this helps.
Good luck!
Anja
I agreed with Refik and Peter. Room temperature is also very significant in post operative management and care.
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