In my hands, surgical technique and highest possible aseptic standard was the solution to the issues you are seeing.

Technique:

The Day 0 image shows several clear spots of subdural bleeding. Avoiding these takes practice, but in my experience is key. In addition, there appears to be some cloudiness in the bottom part of the window. Are you sure you are removing all the bone from the craniotomy? I found it is very easy to leave a think layer in parts of the craniotomy, which could then be a starting point for regrowth. One crutch could be testing that the bone is gone by *carefully* probing areas that you are uncertain about with a *blunt* forceps tip. Alternatively, you could skim the surface of the dura with a blunt forceps (e.g. #3) to remove any leftover bone layers.

Day 2 looks like a vessel ruptured. It is hard to say for sure, but my impression is that this is epidural bleeding. Once a window looks like this, it is unlikely to improve again.

Having said all of the above, windows do tend drop in quality for a few days early on and tend to recover, but if I saw that happen, it never got as bad as what you are showing in your image series.

All of this takes practice and chronic imaging is much more sensitive to minor mistakes than acute imaging. Don't let yourself get discouraged!

Aseptic standard:

Ideally, you should get a clean and healthy window without the need for antibiotics. Similar to cell culture work, antibiotics can cover up mistakes in aseptic technique, without eliminating all the consequences. Once your aseptic technique is perfect, adding antibiotics for good measure is an option, but prior to that, I would recommend dropping it. Potentially get your institutional vet to look over your shoulder during the procedure. Not all the suggestions from the vet will be compatible with your experiment, but there will certainly be some very useful advice to be had.

Good luck!

Thanks for your quick (and very detailed) reply! You've given me plenty to think about. I hadn't previously considered the possibility that a layer of skull was being left behind, as the skull flap seems to break away fairly cleanly in my experience, and the underlying tissue seems to flex in a way I would expect of meninges/brain tissue alone. Is it possible for a layer of skull to be thin enough to resemble the dura in flexibility? In any case, I can certainly try pulling away the skull edges around the window and see if that helps prevent the regrowth.

You also mentioned that there appears to be a vessel rupture in one of the images (I presume you meant day 3, the third image down). Do you know what might be causing this? I've seen some version of it in all my windows so far - it always occurs suddenly after the window has been clear for a day or two, with varying degrees of severity. It seems to be slightly less severe when I include an agarose layer between the coverslip and dura (to stabilize/cushion the brain for imaging), so I suspect it's caused by some kind of trauma, but I'm not sure how that would occur.

To be clear: I wouldn't anticipate the entire craniotomy to have a thin layer of bone. If the tissue flexes locally, rather than as a larger area, when probed, then I would assume all bone has been removed in that area. In my experience, it tended to be a thin layer in a smallish area (never more than about 20% of the craniotomy, due to the way the bone comes away. But don't fixate on this bit - I might well be wrong.

Avoiding trauma during the actual craniotomy is key. My recommendation when training students was always to slowly thin the bone around a central island, while keeping everything damp with ACSF or Ringer's. The thinner the bone gets, the more important it is to keep it damp. Once you find you are thin enough to remove the bone in the center, put some ACSF or Ringer's onto the entire area and let it sit for 5 to 10 minutes. The bone now comes off much easier without sticking to the dura.

Regarding the ruptured vessel, I meant day 3 indeed. It is difficult to say what caused it. It isn't something I saw very often.

Agarose under the glass is a problem for longer experiments, as it can become turbid. I have also heard people mention they saw some vascularization.

Ok, good to know. From your recommendations it sounds like my basic technique is correct, but I'll need to figure out what's causing the blood vessel rupturing. Interestingly, I dissected a 4-week post-surgery window yesterday (completely occluded with a thick membrane) and discovered what appeared to be a membrane coming from beneath the cut edges of the skin on one side, and stretching over across the window. Maybe the problem is incomplete removal of the periosteum? It would certainly account for some of my observations.

In any case: Thank you again for all your help!

That is an interesting finding. It might indeed be a problem. How do you removed the periosteum?

I was taught to scrape the bone with a scalpel blade to remove all tissue from the bone. This worked quite well, but it did require a bit of attention to not apply too much pressure during the scraping.

I also know of people who would apply H2O2 to the bone as part of the cleaning process.

Besides the importrance of clean window without bleeding at window installation time as stated, key to increase of long-term clarity is securing a coverslip plug and applying some pressure to replace skull surface. See https://www.nature.com/articles/nprot.2014.165

for detailed protocol on this.

Hello Thomas Murphy and Christian Wilms ,

Thank you for the information provided above. I have a quick question for you, do you think it is possible to do acute imaging of the cortex? Meaning, would it work to do imaging right after the craniotomy? And in that case, would you say that a coverslip is necessary or could I put my objective right over the cortex ?

Thanks in advance for your responses!

Hi Sebastian,

That is absolutely possible. My main imaging work was actually acute in vivo. Details will depend on your exact experiment. I have worked in neocortex with and without a coverslip. The limiting factor is pulsation. If imaging isn't clear enough, you might need to remove the dura - if you do, you will almost certainly need a coverslip.

You will want to check with your veterinary team and the respective legislation, in case you want to do the acute imaging in an awake animal, as recovery times might not be sufficient.

Good luck!

Thank you for your response Christian Wilms !

I am planning on doing acute imagery on anesthetized mice.

I will try both ways, with and without removing the dura.

I looked at your papers, may I just ask you, in your Nat. Comm, what do you mean exactly when you write that you glued the coverslip to the imaging chamber?

I mounted a headplate to the skull of the mouse. This had a round opening in the centre, and flat surface surrounding that. I glued the coverslip to that flat surface (inner opening was about 3 mm, the coverslip 4 mm diameter. That gave me an even surface to glue to. As this was acute, I was able to use agarose in between.

Hello and greeting Prof/ Christian Wilms and Thomas Murphy

Thank you for your valuable informations

I try to do cranial window to study the changes in neurons after stroke model through using two photon imaging , I used YFP mice and I waiting 7 days before imaging it according to these papers( Skull optical clearing window for in vivo imaging of the mouse cortex at synaptic resolution, Imaging Neural Activity in the Primary Somatosensory Cortex Using Thy1-GCaMP6s Transgenic Mice) to get a high quality image but actually the image not good and i couldn't recognized the neural cell

so can you help me to understand the reason and give me some advise such it is better to do stroke model (photothrombosis stroke) after or before cranial window

I attached my two photon images

I want to know the suitable dose of dexamethasone and carprofen for 20 gm mouse and the dilution of both dexamethasone and carprofen Thomas Murphy

Typical dose for Dexamethasone is 2 mg/kg intramuscular, and for Carprofen is 5 mg/kg intraperitoneal (See these papers: http://cshprotocols.cshlp.org/content/2012/6/pdb.prot069617, Article Chronic cranial window with access port for repeated cellula...

Incidentally, I strongly recommend the protocol by Holtmaat et al 2012 (the first link above), as well as their earlier nature protocol from 2009 ( https://www.nature.com/articles/nprot.2009.89 ). They are highly detailed and informative resources for this kind of surgery.

It's difficult to tell what is wrong with your windows based on the images you provided - a sample z-stack may be more informative. It does seem like you may have skull regrowth partially covering the window, though. Is the window visibly occluded when you examine it by eye?

Thomas Murphy Thank you for your response , when i examine the window by eye or normal microscope it is visible and i can see the blood vessels very well

Thomas Murphy I want to do photothrombosis stroke on brain ,so what is your suggestion about it (do it before cranial window or after)

Unfortunately I don't have any direct experience with stroke models, so I can't offer any particular expertise with regard to your model. More importantly though, it probably depends largely on what questions you want to ask. I would imagine it makes the most sense to induce the stroke after window implant, since this (1) allows for baseline images if you're interested in a time course of changes in a specific neuron group, and (2) doesn't produce additional inflammation before the implant, which could impair window recovery.

Thank you very much Thomas Murphy

I am facing the same problem dealing with chronic window in the mouse auditory cortex. It is getting hazy in areas of interest and barring me from continuous imaging. I don't think it is due to the skull growth, but, due to the oozing out of fluid from the brain - which is mostly happen due the hitting of the head plate on the cage wall (or they have tried to pull if off in a organised way).

Repeating the antibiotic and dexamethasone sometimes recovers the window for further imaging but the chance of success is minor.

I think strong anti-inflammatory drugs (with proper dose) could help to a fast recovery. However, there may be varied conditions for the hazy glass window, as I had previous experience of grown fungus/ candidiasis (possible and not confirmed by culture study) below the glass window.

As, a preventive measure, I focus in the sterile condition of the surgical table and tools - this has certainly increased the transparent window life from 5-6 days to more than two weeks.

Hello, I'm having a similar issue. Could you figure out so far how to improve the stability of a clear window? Thanks