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A Craniotomy Surgery Procedure for Chronic Brain Imaging

1, 1

1Department of Neurology, University of California, Los Angeles

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    This video and protocol demonstrate how to implant a glass-covered cranial window in rodents. These preparations can be used for chronic in vivo two-photon imaging of the neocortex over time scales of months. It may also be used for other types of imaging, including optical intrinsic signal imaging.

    Date Published: 2/15/2008, Issue 12; doi: 10.3791/680

    Cite this Article

    Mostany, R., Portera-Cailliau, C. A Craniotomy Surgery Procedure for Chronic Brain Imaging. J. Vis. Exp. (12), e680, doi:10.3791/680 (2008).


    Imaging techniques are becoming increasingly important in the study brain function. Among them, two-photon laser scanning microscopy has emerged as an extremely useful method, because it allows the study of the live intact brain. With appropriate preparations, this technique allows the observation of the same cortical area chronically, from minutes to months. In this video, we show a preparation for chronic in vivo imaging of the brain using two-photon microscopy. This technique was initially pioneered by Dr. Karel Svoboda, who is now a Howard Hughes Medical Institute Investigator at Janelia Farm. Preparations like the one shown here can be used for imaging of neocortical structure (e.g., dendritic and axonal dynamics), to record neuronal activity using calcium-sensitive dyes, to image cortical blood flow dynamics, or for intrinsic optical imaging studies. Deep imaging of the neocortex is possible with optimal cranial window surgeries. Operating under the most sterile conditions possible to avoid infections, together with using extreme care to do not damage the dura mater during the surgery, will result in successful and long-lasting glass-covered cranial windows.


    1. Anesthetize mice with isoflurane (4% for induction, 1.5-2% for surgery) using IACUC approved procedures. It is important that tail and/or toe pinches are used in order to ensure the animal is fully sedated.
    2. Using a rodent trimmer, shave the hair from the back of the neck up to the eyes.
    3. Place the mouse in a stereotaxic frame, over a surgery water re-circulating blanket. Firmly secure the head with ear bars.
    4. Apply eye ointment, in order to prevent the animal's eye from drying out.
    5. Administer, subcutaneously, Dexamethasone (0.2 mg/Kg) and Carprofen (5 mg/Kg) to prevent swelling of the brain and/or an inflammatory response, respectively.
    6. Before beginning the surgery, sterilize the operating area by wiping skin with three alternating swipes of 70% alcohol and betadine.
    7. All surgical instruments have been pre-sterilized using a glass bead sterilizer. Using scissors that have been sterilized with ethanol, remove the skin over the top of the skull, starting with a horizontal cut all along the base of the head, followed by two cuts in the rostral direction, almost reaching the eyelids, then two oblique cuts that converge at the midline.
    8. A drop of lidocaine + epinephrine solution is applied at this point onto the periosteum to avoid excessive bleeding or pain. With a scalpel, retract the periosteum to the edges of the skull.  Also, lightly retract the musculature of the back of the neck.
    9. Gently scrape the entire exposed area of the skull with the scalpel to create a dry surface. This is very important, as it will allow the glue to adhere better when applied later.
    10. Once an imaging site has been chosen, one is ready to create the cranial window. First, gently "draw" a circle of about 4 mm in diameter with the pneumatic dental drill.
    11. After a slight drilling, apply lidocaine + epinephrine solution again onto the skull surface. Stop the drilling when a very thin layer of bone is left. By pushing gently on the center of the craniotomy to feel how it gives way, one can usually know that this stage is reached.
    12. Under a drop of saline and taking advantage of the bone trabeculae - the spongy structure of the bone - lift away the craniotomy from the skull with very thin tip forceps. The saline is important, as it will help lift up the skull and prevent bleeding of the dura.
    13. Apply Gelfoam that has been previously soaked in saline to the dura mater to stop any small bleeding that occassionally occurs when the skull is removed.
    14. After drying the dura mater surface and ensuring that there is no bleeding, gently lay a sterile 5 mm glass cover slip on top of the dura mater. (Note: other groups also place a drop of low melting point agarose (1.2%) over the dura and put the coverslip on top of the agar).
    15. Apply a drop of cyanocrylate-based glue to the opposite hemisphere on the skull. With the help of a needle, gently apply the glue all around the window while being careful not to put it under the glass. Glue can now be applied in a thin layer over the entire surface of the skull.
    16. Once the glue has dried, mix dental acrylic and apply it throughout the skull surface, covering also a small rim of the cover slip, to secure it.
    17. After securing the cover slip, make a small well around the window with dental acrylic. Also, embed a titanium bar in the dental acrylic. This bar will later be used to attach the mouse securely on to the stage of the microscope for imaging. It is important to ensure that the bar is level, so that it is parallel with the cranial window. Placing a piece of paper under the bar can allow the bar to remain level while the acrylic hardens.
    18. The dental acrylic is allowed to cure (harden) for 10 minutes, by which time the titanium bar is fixed in place. Place the animal in a warm cage until it recovers.
    19. After recovery from anesthesia, the animal can be imaged on the same day.

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    As we have shown in the video and in the supplementary figures, the cranial window preparation, combined with the use of two-photon microscopy, is a very powerful tool to study in vivo the structure and function of the neocortex. The technique requires rigorous training to become familiar with the relevant anatomy and the fine surgical procedures and skills that this preparation requires. Only pristine surgeries can be used for chronic imaging. If the dura is manipulated excessively or punctured, the preparation should not be used for imaging.

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    Name Type Company Catalog Number Comments
    Carprofen (Rimadyl) Drug Pfizer Pharma GmbH
    Isoflurane (Aerrane) Surgery Baxter Internationl Inc.
    Dexamethasone Drug Baxter Internationl Inc.
    Ortho-Jet Powder Reagent LANG To be mixed with the acrylic
    Jet-Acrylic Liquid Reagent LANG To be mixed with Ortho-Jet Powder
    Round Glass Cover Slip Tool Electron Microscopy Sciences 72195-05 5 mm diameter
    Gelfoam Surgery Pharmacia Corporation (Pfizer)
    Titanium bars are custom-made


    1. Svoboda, K., Denk, W., Kleinfeld, D., Tank, D. W. In vivo dendritic calcium dynamics in neocortical pyramidal neurons. Nature. 85, 161-165 (1997).
    2. Lendvai, B., Stern, E. A., Chen, B., Svoboda, K. Experience-dependent plasticity of dendritic spines in the developing rat barrel cortex in vivo. Nature. 404, 876-881 (2000).
    3. Trachtenberg, J. T., Chen, B., Knott, G. W., Feng, G., Sanes, J. R., Welker, E., Svoboda, K. Long-term in vivo imaging of experience-dependent synaptic plasticity in adult cortex. Nature. 420, 788-794 (2002).
    4. Portera-Cailliau, C., Weimer, R. M., DePaola, V., Caroni, P., Svoboda, K. Diverse modes of axon elaboration in the developing neocortex. PLoS Biol. 3, (2005).
    5. Holtmaat, A., Wilbrecht, L., Knott, G. W., Welker, E., Svoboda, K. Experience-dependent and cell-type-specific spine growth in the neocortex. Nature. 441, 979-983 (2006).



    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

    Posted by: AnonymousMarch 25, 2008, 8:05 AM

    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 luckRicardo Mostany 

    Posted by: AnonymousMarch 25, 2008, 1:49 PM

    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

    Posted by: AnonymousFebruary 11, 2009, 8:10 PM

    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

    Posted by: AnonymousApril 17, 2009, 1:38 PM

    Hi, Could you provide me with the manufacturer and model number of the pneumatic drill that you use?   Thanks a lot,   Dan

    Posted by: AnonymousJune 17, 2008, 8:06 PM

    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.      

    Posted by: AnonymousJune 17, 2008, 9:48 PM

    Hi, Could you please tell me the size of the drill bit that you use during craniotomy? Thank you

    Posted by: paul z.October 21, 2014, 4:31 PM

    Hello, Could you please tell me the dimensions of your titanium bar, and also its weight? Thanks for your help. James T. Russell

    Posted by: AnonymousJuly 3, 2008, 1:19 PM

    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

    Posted by: AnonymousJuly 7, 2008, 2:20 PM

    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

    Posted by: AnonymousJuly 9, 2008, 6:13 AM


    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

    Posted by: AnonymousJuly 9, 2008, 5:35 PM

    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

    Posted by: AnonymousJuly 11, 2008, 3:30 PM

    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    

    Posted by: AnonymousSeptember 17, 2008, 7:20 AM

    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

    Posted by: AnonymousSeptember 17, 2008, 1:36 PM

    Hi Ricardo and Ralph, We use a UV acrylic from Pentron Dental that sets in 10 seconds under UV light. Tom

    Posted by: AnonymousNovember 1, 2008, 11:17 PM

    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 micrŒlectrodes 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

    Posted by: AnonymousSeptember 18, 2008, 4:23 AM

    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.  

    Posted by: AnonymousSeptember 18, 2008, 3:26 PM

    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.

    Posted by: AnonymousMarch 18, 2009, 1:15 PM

    Hi very nice video! Raquel Revilla-Sanchez Tufts University

    Posted by: AnonymousSeptember 27, 2008, 7:11 PM

    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

    Posted by: AnonymousOctober 26, 2008, 11:57 PM

    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

    Posted by: AnonymousOctober 27, 2008, 1:28 AM

    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
    Albert Einstein College of Medicine
    Dept. of Anatomy and Structural Biology
    Gruss-Lipper Biophotonic Center

    Posted by: AnonymousFebruary 9, 2009, 1:08 PM

    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

    Posted by: AnonymousFebruary 10, 2009, 3:05 PM

    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;²0-1515).  the gel dŒsn'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

    Posted by: AnonymousFebruary 20, 2009, 11:32 AM

    Hello Jason,   Thanks for your suggestion about the gel-like glue. We will try it. Krazyglue also dŒs 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  

    Posted by: AnonymousFebruary 24, 2009, 1:52 PM

    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, dŒs that indicate an obvious error I am making? I would apprecite any input you can give me.   thank you, cg

    Posted by: AnonymousFebruary 20, 2009, 5:14 PM

    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

    Posted by: AnonymousFebruary 24, 2009, 2:02 PM

    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) DŒs 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. DŒs it has some thing to do with the problem we are facing? Any comment is appreciated. Thanks.

    Posted by: AnonymousFebruary 25, 2009, 8:12 AM

    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.

    Posted by: AnonymousMarch 18, 2009, 1:10 PM

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

    Posted by: AnonymousMarch 9, 2009, 9:50 AM

    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

    Posted by: AnonymousApril 16, 2009, 4:42 PM

    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

    Posted by: AnonymousApril 17, 2009, 11:44 AM

    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

    Posted by: AnonymousApril 28, 2009, 12:04 AM

    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

    Posted by: AnonymousMay 6, 2009, 3:27 AM

    Hi Ricardo
    Thank you very much for your share.Could you tell me where can I get the titanium bar?
    Thank you.
    Fei Li

    Posted by: AnonymousAugust 25, 2009, 7:07 AM

    Hello Fei,

    Those titanium bars are custom made. We got them from the machine shop here at UCLA.


    Posted by: AnonymousAugust 25, 2009, 1:44 PM

    Recently I'm trying to do the same surgery.Thank you very much for giving such a video.But it was difficult to get the Dexamethasone and Carprofen. Could you tell me where can I get it? Thank you.
    Fei li

    Posted by: AnonymousAugust 31, 2009, 3:38 AM

    Hello Fei,
    Here at UCLA we get the dexamethasone from the Hospital (our lab is part of the School of Medicine) and the carprofen from the pharmacy of the veterinary service.

    Posted by: AnonymousSeptember 10, 2009, 2:15 AM

    very good

    Posted by: john d.November 6, 2009, 12:38 PM

    Thanks for the detailed video! I've also been struggling with keeping my windows clear, and noticed that you guys don't seem to put anything on the dura after you clean it, right before putting the coverslip on. Is there anything at all under the coverslip, like ACSF or saline or PBS, or just relatively dry dura contacting the middle of the coverslip, and then just air, then the acrylic around the far edges of the coverslip? Or do you put a little pressure in order to gently and slightly flatten the brain/dura so that it all makes contact with the glass and there is no air left?

    Thanks very much!

    Posted by: V C.May 14, 2010, 12:06 PM

    As you guessed, there is nothing between the dura and the cover glass, just relatively moistened/dry dura. The Krazyglue helps to seal the craniotomy and the slight pressure on the coverslip makes the dura to be flat and no air gets trapped between the dura and the craniotomy.

    Posted by: AnonymousJune 1, 2010, 6:20 PM

    Hi, would you please send me information on the heated blanket you are using during surgery? Many thanks, Ute

    Posted by: Ute F.August 13, 2010, 10:44 AM

    Hello Ute
    We use a water circulating pump Gaymar T/Pump TP-500. It's sold from many different sources.
    Hope this helps

    Posted by: AnonymousAugust 13, 2010, 2:49 PM

    Thanks for getting back to me. I do have a pump, but I cannot find a suitable (small, thin) blanket for it. I want to use it in combination with a mouse stereotaxic apparatus. Where did you buy yours? Thanks, Ute

    Posted by: Ute F.August 16, 2010, 12:10 PM

    We bought the standard ones and fold them, although there are smaller sizes. We ordered directly from Gaymar

    Posted by: AnonymousAugust 17, 2010, 1:18 PM

    Thank you, Ricardo! Ute

    Posted by: Ute F.August 17, 2010, 1:56 PM

    Hello that's a great video, I will use multiphoton microscopy during several weeks,how I could fix the metal bar to the microscope's table.

    Posted by: AnonymousMay 27, 2011, 11:24 AM

    The bar has a tapped hole so you can use a screw to attach the bar to a holder


    Posted by: AnonymousMay 27, 2011, 2:45 PM

    The titanium bar has a tapped hole so you can use a screw to attach the bar to a holder


    Posted by: AnonymousMay 27, 2011, 2:45 PM

    Ok, just another thing, what kind of holder you use? You developed the holder? or, could you provide me a serial number? Thanks

    Posted by: AnonymousMay 31, 2011, 9:49 AM

    The holder is custom made


    Posted by: AnonymousMay 31, 2011, 2:13 PM

    I had a question about your video itself; what kind of camera and microscope setup did you use to take the video of the surgical procedure?

    Posted by: AnonymousJune 13, 2011, 5:00 PM

    I don't know. JoVE staff came with their equipment and filmed the video.

    Posted by: AnonymousJune 14, 2011, 3:23 PM

    Hello, the work that you carried out is incredible, I have a question, how you conect the vaporizer to mouse's nose? did you use O²?

    Posted by: AnonymousSeptember 22, 2011, 5:24 PM

    We use plastic tubing and then a rubber dropper (like you would sue for Pasteur pipettes) that we cut on both ends to fit over the nose. You can see ti pretty well on the video. And yes, we use oxygen at 0.5 Liters/min
    Good luck!

    Posted by: AnonymousSeptember 22, 2011, 5:32 PM

    Thank you for this useful video. I have tried to do this open-skull surgery these days and by now I have faced some problems that I want to consult you. First, could you please tell me the dimensions of your cover glass? I used a cover glass 5mm in diameter but it seems a little bit large. Second, during my surgery the dental cement always get under the cover glass, could you please tell me how to circumvent this? Third, after my surgery, the animals cannot be imaged because something emerged under the glass window and obscured the idle imaged area, I guess it results from tissue proliferation. Were you confronted with this and how do you solved?
    That's all, Thank you very much!
    Best wishes!

    Posted by: AnonymousOctober 11, 2011, 9:45 PM

    Thanks. We use 5 and 3mm cover glasses, depending on the size and the position of the craniotomy. About the dental cement, if you watch the whole video and read the protocol, you will see that we use cyanocrylate glue (super-glue) to first glue the cover glass. The glue will avoid the cement to get under the glass.
    Regarding bad quality of the windows, it could be due to several factors: damage to the dura, bone growing back, infections... Wash exhaustively the surface of the brain with saline before you close the window, that will help.
    Best of luck

    Posted by: AnonymousOctober 12, 2011, 2:35 PM

    Thank you very much for your reply. I tried to glue the cover glass by super glue, but I met another problem that the cover galss is plane while the "circle" around the open area is not so that the cover glass cannot glue the whole "circle", maybe half the circle, then how to do with the other half . One more question is when I tried to use super glue to bond the cover glass, the super glue might also come under the cover glass. I'm bothered with these problems for a long time and I wish to work out them as soon as possible. Thank you very much again for your useful reply!
    Best wishes !

    Posted by: AnonymousOctober 12, 2011, 9:33 PM

    There is no problem if a little bit of glue come under the cover glass just around the window.It will seal the window and glue the cover glass.

    Posted by: AnonymousOctober 14, 2011, 2:35 PM

    Thank you very much for your previous reply. Could you please tell me where to buy the dental cement you used and which product catalog it is?
    Best wishes!

    Posted by: AnonymousOctober 26, 2011, 11:21 PM

    We use this: Ortho-Jet Acrylic Powder clear (1330CLR) + Ortho-Jet Acrylic Liquid clear (1303CLR) from Lang Dental

    Posted by: AnonymousOctober 28, 2011, 5:57 PM

    We use this: Ortho-Jet Acrylic Powder clear (1330CLR) + Ortho-Jet Acrylic Liquid clear (1303CLR) from Lang Dental

    Posted by: AnonymousOctober 28, 2011, 5:56 PM

    Thank you very much for your generous experience sharing. I really appreciate your replies before.
    I have tried to do the surgery following your vedio these days. The images taken immediately after the surgery were ideal, but the images taken after recovery for ²1d seemed strange. Instead of clear neuronal structures, in the image were some bright plaques which were between the cover glass and the expected neuronal structures. And under the bright plaques no or only faint fluorescence of neuronal structures could be detected.
    I was confused by these images, so I ask for your help again. I guess that it resulted from infammatory reactions following the surgery. What do you think about it? I noticed that you and several other investigators chose to inject carprofen to prevent the inflammatory reactions. I wonder why you choose carprofen. Can I use other similar drugs to take place of it because it seems hard for me to purchase carprofen.

    Posted by: AnonymousNovember 21, 2011, 4:05 AM

    Hello Sophy,
    I can't tell you exactly what is causing the appearance of such bright plaques but obviously it is a bad symptom. Sometimes it is debris from dying cells that macrophages internalized, but it could also be bone. If you want, you could send me an image (stack) so I could have a look. I always inject carprofen (you can use any other non-steroidal anti-inflammatory drug) and dexamethasone (a glucocorticoid) to avoid swelling and/or edema. If the brain is bulging a lot when you remove the skull, the pressure against the coverglass could damage the brain. With these drugs the pressure will be less and the chances that your window stays in good condition for the imaging increase.
    By the way, sorry for the late response.

    Posted by: AnonymousJanuary 23, 2012, 8:30 PM

    Hi, Thanks for the nice video. we are trying to visualize the implanted intracranial fluorescent tumor on the mouse brain through two photon imaging but having hard time. The depth of the injection is 1mm. Is that two deep to view? our window is clear not opaqe. Please suggest.

    Posted by: AnonymousJanuary 23, 2012, 12:56 PM

    Hello Bhas,
    Even if the window is clear, 1mm it is probably too deep (there are exceptions, obviously). I don't know the fluorophore you are using, but if you want to go so deep, you should chose a red one (the longer the wavelength, the deeper you can reach). Another suggestion is to incorporate to your system an OPO (optical parametric oscillator). Check this paper from Kobat D et al ²011 (In vivo two-photon microscopy to 1.6-mm depth in mouse cortex. Journal of Biomedical Optics 16(10), 106014 (October ²011))
    Hope this helps

    Posted by: AnonymousJanuary 23, 2012, 8:50 PM

    Hello Ricardo,

    Thanks much for the video. I was wondering about your comment regarding using agarose before sealing off the craniotomy. Have you guys tried using it? How do you keep the agarose in solution form without it clumping? The microwave method used for preparing agarose usually leads to it forming a gel. This is probably very trivial but I have a hard time trying to figure this out. We are trying to make a cranial window for superior colliculus in rats which is a mid brain structure. We remove the overlying cortex and I am yet to figure out how to make a cranial window. And I am running acute experiments for now in case you were wondering.

    Thank you!

    Posted by: AnonymousApril 8, 2012, 7:51 PM

    I did try it at the very early stages of my learning and I always struggled with it. We used low melting agarose, so once boiled, the temperature can drop a lot (so you don't burn the surface of the brain) before solidifying. Good luck.

    Posted by: AnonymousApril 9, 2012, 3:31 PM

    Hello Ricardo,

    Thanks for the reply. Was the issue maintaining it as a solution ?

    Posted by: AnonymousApril 9, 2012, 3:48 PM

    Right, while in the beaker and still warm, the agarose stays as a liquid, but as soon as you use a dropper to apply it, solidifies because of the change in temperature, so you have to be really fast. It's doable, so you just need to practice. I managed to skip it for my preps, and they work great.

    Posted by: AnonymousApril 9, 2012, 4:39 PM

    Hi Ricardo,
    Thanks for the excellent video. I have been doing craniotomies for a bit now and have improved my technique such that I can perform a clean surgery without damaging the dura or the brain. However, I still find that after a few days, there is a very vascular membrane that seems to grow over the brain and parts of the surrounding skull. The other possibility is that this membrane is simply a swollen, inflamed meninges. I was wondering if you have every had this issue and what you do to address it.

    Posted by: David B.July 22, 2012, 2:38 PM

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