Hello,

I'm a PhD student from Florence, Italy, and I wuold like to know what is the concentration of the epinephrine/lidocaine solution that you use.

Thanks,

Anna Letizia Allegra

Hello Anna,

We use a Lidocaine HCl 1% and Epinephrine 1:100,000 solution. This solution is comercially available from several companies. Ours is from Hospira, Inc.

Hope this helps you. Good luck

Ricardo Mostany

Hi,

thank you for the very useful video! What do you think about the use of lidocaine-epinephrin in acute, as opposed to chronic, preparations? I.e. do you think the solution applied to the skull during surgery could have effects on neuronal activity imaged soon afterwards - like an hour or two later?

Best,

Zuzanna Piwkowska

Hello Zuzanna

The purpose of the lidocaine-epinephrin is double; to reduce pain sensation (from the incision in the skin) and to reduce bleeding from skull while drilling. I apply it very soon in the surgery: before removing the periosteum and sometimes after drilling for a little while the skull (when starting to drill the spongy bone). My guess is that it shouldn't have any effect on neuronal activity. I don't know what could happen in case you apply it later on during the surgery, when the dura is accesible (and even with some minimal nicks).

Good luck

Ricardo

Hi,

Could you provide me with the manufacturer and model number of the pneumatic drill that you use?

Thanks a lot,

Dan

Hi,

We bought our pneumatic drill from Henry Schein:

Traditional Handpiece with Power Lever Catalogue # 7726063.

Laboratory Handpiece Control Kit:  Cat # 6427057.

If you can't find the second item, you can google "Laboratory Handpiece Control Kit" and a number of other vendors will come up.

Hello,

Could you please tell me the dimensions of your titanium bar, and also its weight?

Thanks for your help.

James T. Russell

Hello James,

The dimensions of the titanium bar are these (all in inches):

0.125x0.375x0.05

The holes are two #2-56 tapped holes, symmetrically located at both edges of the bar. Only one hole is necessary but the other one helps to attach (embed) the Ti bar to the acrylic.

The weight is around 130mg

Good luck

Ricardo Mostany

Thanks for the useful video. We are following a similar procedure which differs very slightly and works great without any flaw. My question here is not about the glass window but about a thin skull chronic preparation. I tried it a few times…It is great on Day 1 but become opaque on day 2. You can’t see on day 2 as clearly as you did on day 1 any vasculature or dendrites through the window? What is your experience in thin skull chronic preparation? Do U have any comments?

Thanks

MTR

Hello,

We haven't done that preparation. We had heard about that disadvantage before. You have to do the thinning repeteadly because the bone grows back and makes difficult to do the imaging. I don't know if in two consecutive days this is so critical.

Good luck

Thanks for your response. I think in addition to growth of bone (which may take some time to happen) there is wound healing process that starts in the bone which reduces the transparency. I will be trying more mice though. Thanks.

MTR

test

Hello Ricardo,

I saw the video and I think this helps a lot of people by seeing how surgery is done. Very nice.

Just a tip about using acrylic cement. We use "GC Fuji Plus" a glass ionomer luting dental cement (with supportscrews in rats) which comes in capsules. It hardens very fast ( 1-2 min.) so you don't have to wait but you can almost instandly continue your surgery. I don't know if it mounts very strong on cyanocrylate-based glue or maybe directly onto the skull. You'll have to test that.

regards

Ralph

Hello Ralphs,

Thanks a lot for your comment. That dental cement could be helpfull too. I will let you know if I have a chance to try it and give you my feedback.

Best

Ricardo

Hi Ricardo and Ralph,

We use a UV acrylic from Pentron Dental that sets in 10 seconds under UV light.

Tom

Hello,

The video is helpful for me. We are planning to conduct similar chronic craniotomy surgery on rats. The difference is that we want to drill three 1mm diameter holes and implant microelectrodes into the brain. So use harmless glue is important for us.

My question is why not use acrylic cement directly and is the cyanocrylate-based glue you used poisonous to the brain?

Hello,

The video is wery helpful for me. We are planning to conduct similar chronic craniotomy surgery on rats. The difference is that we want to drill three 1mm diameter holes and implant microelectrodes into the brain. So use harmless glue is important for us.

My question is why not use acrylic cement directly and is the cyanocrylate-based glue you used poisonous to the brain?

Best,

Xuan

Hi,

Acrylic cement needs more time to cure and secure the window than these cyanocrylate glues. So the risk of getting glue or acrylic under the cover glass diminishes. That's why I rather the glue. In your case, I don't know how this will affect.

Regarding the toxicity of these glues. If you do the surgery properly, only the very outer edges of the window will be in contact with the glue. Anyhow, these cyanocrylate based glues are also used in open wounds to keep the edges together (e.g. Nexaband). I have never observed damaged tissue in the window because of the glue, even after perfussion and histological studies.

Regards

Ricardo.

i'd also add that it is important to create an "air-tight" seal around the window - the acrylic is likely to be porous.  i tried using only cement (dental cement, bone cement) to seal and the windows only last 3-4 weeks, whereas using glue all the way around keeps them good for months and markedly improves success rate.

Hi

very nice video!

Raquel Revilla-Sanchez

Tufts University

Hi,

Thanks for the useful video. I had taken great advantage from your information.

Though, I have one question.

Sometimes, even with great caution, there comes bleeding when I take off the skull.

It occurs at the center of the craniotomy region as well as at the edge.

Can you please comment on this kind of problem?

I have put epinephrine/lidocaine solution and also saline just before taking off the skull.

Is there any delicate way to lift it up?

Thanks,

Jinho Kim

I usually drill until the skull is thin enough to be cracking.  I then wet the skull with gelfoam soaked with cortex buffer and wait a bit.   Finally I use fine forceps and try and lift off at a place with few vessels.  I push the forcep tip into the thickness of the skull and try and not go under it and then lift off. 

I hope this helps,

Peyman Golshani

Hi guys,

that's a really nice and clean-looking procedure. I think we will post one of these videos too.

Could you give me ordering information for Gelfoam? Do you use the powder or sponges, what's the catalog number, where do you order from and did they ever ask you for some paperwork (wholesalers told me you need a prescription)?

Thanks in advance

Bojana Gligorijevic, PhD
1-718-678-1130
Albert Einstein College of Medicine
Dept. of Anatomy and Structural Biology
Gruss-Lipper Biophotonic Center

Hi Bojana. Ethicon Surgifoam Absorbable Gelatin Sponge size 100 COMPRESSED (12.5cmx8cmx2mm) from Owens & Minor ~$140 Tara Spires-Jones, DPhil Instructor, Harvard Medical School MassGeneral institute for Neurodegenerative Disease 114 16th St, Charlestown, MA 02129

hi,

thank you for posting this "article"...after many different approaches, this technique has worked the best.  one comment that may be helpful to others - instead of "liquid" glue, i started using one with a thicker gel-like consistency (Loctite 454 gel; http://www.mcmelectronics.com/product/20-1515).  the gel doesn't flow at all, which helped in my situation.  i could not hold and press the window very hard during glue application because i was working in young mice with very soft skulls.  you can apply it by using a 30g needle as a 'palette knife' to build it up around the window, creating a seal.  how do you prevent the liquid glue from lurching under the window?

jason coleman

Hello Jason,

Thanks for your suggestion about the gel-like glue. We will try it. Krazyglue also does the trick (If the tube has been open for several days, the glue gets thicker, and easier to apply)

Regarding your question, the few times I have done surgeries on young animals, I have used agarose or I have applied the glue very carefully, holding and slightly pressing the coverglass.

Ricardo

Thank you for posting the video.  I have another question to add to the forum...

Do you ever have any evidence of seizures during or following the procedure, and if not, does that indicate an obvious error I am making?

I would apprecite any input you can give me.

thank you,

cg

Hello Cristin,

Isofluorane sometimes elicits seizures in mice (rarely). I don't know if that is the anesthetic you use.

Also, if you are pressing the brain too much (maybe because of excesive swelling), you may be stopping the blood flow in the superficial brain arteries (MCA, ACA, the main blood supplier in the cerebral cortex) and that can cause seizures.

Hope this helps

Ricardo

I have few questions regarding chronic imaging in this preparation. Window in our mice stays clear and transparent without any infection over a period months after we made them. Nonetheless,

1) The number of dendrites that can be imaged as well as image quality decreases as the time passes.

2) We need to use as much 5-10 fold higher laser power to acquire images after 2 weeks as compared to whatever power we used on day 0? After 2 weeks of window preparation it reaches a point where we need an extent of laser power that damages the brain before we can acquire some images???

3) Does repeated imaging damages the brain when powers of less then 25 mW at 925 nm used? How many imaging sessions can be done from the same window in a typical situation over a period of 30 days? What are the factors that we need to consider for a glass window preparation that gives good images over a period of month.

4) We know some wound healing process starts and covers the brain within few days of the window preparation at least in some animals. Some labs report a sucess rate of 30-50% (1 in 2 mice or 1 in 3 mice) for long term imaging. I mean the preparation staying imagebale on chronic basis over a perid of 1-2 months? What is typical success rate?

5) We do not administer dexamethasone or antibiotic to mice as we feel that may interfere with the study. Does it has some thing to do with the problem we are facing?

Any comment is appreciated. Thanks.

one note - after almost 2 years of trying for consistently good windows, i found the pre-op administration of dexamethasone and carprofen to be crucial (in addition to following this entire protocol) for improving success rate of keeping clear windows for weeks to months (e.g. <10% success prior to use; >90% while using).  i have spoken with another 2-p expert that reports a similar experience and whom always uses dex pre-op.

I might also suggest that if you use the acrylic without the glue, the acrylic doesn't stick very well to the skull.  It ends up being more like a motile hat that is movable than an anchored, fake skull. 

Thank you for the nice video. As to the pre-op administration of the two drugs, could you tell me how long (hours, minutes or right before the surgery) you administored the drugs.

Thanks

Jinglu

Hello Jinglu,

I inject the animals right before the surgery, both drugs subcutaneously. They are very important to avoid inflamation and edema, that may cause the window to become opaque. If your windows are not clear enough in a regular basis, try to inject carprofen once daily for 3-4 days. Then let the window set for a few day.

Ricardo

Hi Ricardo,

Thank you for the fantastic video! It has been very helpful for me. I am also struggling with window-clouding issue after surgery. I recently tried administering both dexamethasone and carprofen for a one week period. The windows in all of my mice stayed beautiful while they remained on the medications. However, they all clouded within a couple of days after removing removing them from the drugs. Do you think that it is a bad idea to maintain dexamethasone past the peri-operative period?

I am also using both FVB/N and C57 mice. I'm getting the sense that the outcome is slightly better in the C57 mice. Do you have any experience with FVB/N strain?

Thank you very much for any thoughts.

Sincerely,

Tyson

Hi Ricardo,

Thank you for the fantastic video! It has been very helpful for me. I am also struggling with a window-clouding issue after surgery. I recently tried administering both dexamethasone and carprofen for a one week period. The windows in all of my mice stayed beautiful while they remained on the medications. However, they all clouded within a couple of days after stopping the drugs. Do you think that it is a bad idea to maintain dexamethasone past the peri-operative period? Should I try to taper carprofen to avoid re-stimulating a reactive process?

I am using both FVB/N and C57 mice. I'm getting the sense that the outcomes are slightly better in the C57 mice. Do you have any experience with differences between varying strains of mice?

Thank you very much for any thoughts!

Sincerely, 

Tyson