만성 뇌 영상에 대한 Craniotomy 외과 절차

Biology

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Summary

이 비디오 및 프로토콜은 설치류에 유리 커버 두개골 창을 이식하는 방법을 보여줍니다. 이러한 준비는 생체내 개월 시간 단위 동안 네크로 텍스의 2 광자 이미징에서 만성 사용할 수 있습니다. 그것은 광학 내장 신호 이미징을 포함한 이미징의 다른 유형에도 사용할 수 있습니다.

Cite this Article

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

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Abstract

이미징 기술이 연구의 뇌 기능에 점차 중요 해지고 있습니다. 그것이 사는 그대로 두뇌의 연구를 수 있기 때문에 그 중에서도 두 광자 레이저 스캐닝 현미경은 매우 유용한 방법으로 떠오르고있다. 적절한 준비와 함께,이 기술은 몇 분 거리 개월, 만성 같은 피질 영역의 관찰하실 수 있습니다. 이 비디오에서는, 우리는 두 광자 현미경을 사용하여 두뇌의 생체내 이미징에서 만성위한 준비를 보여줍니다. 이 기술은 처음에는 지금 Janelia 팜에서 하워드 휴즈 의학 연구소의 탐정이다 박사 카렐 스보로 개척되었다. 여기에 표시된 것과 같은 준비가 이미지 대뇌 피질의 혈류 역학에, 칼슘에 민감한 염료를 사용하여, 또는 내장 광학 이미징 연구의 연결 활동을 기록하는 neocortical 구조 (예, 돌기 및 axonal 역학)의 영상에 사용될 수 있습니다. 네크로 텍스의 깊은 영상은 최적의 두개골 창 수술 가능합니다. 수술하는 동안 두라 메이터을 손상하지에 극단주의를 사용하여 함께, 감염을 피하기 위해 가능한 한 가장 멸균 조건 하에서 운영, 성공적이고 지속적인 유리 덮인 두개골 Windows에서 발생합니다.

Protocol

  1. IACUC 승인 절차를 사용하여 isoflurane와 쥐 (유도 4 %, 수술을 1.5-2 %)을 마취. 꼬리 및 / 또는 발가락 핀치스이 동물이 완전히 진정되도록하기 위해 사용하는 것이 중요합니다.
  2. 쥐 트리머를 사용하여, 최대 눈 목 뒤쪽에서 머리를 면도.
  3. 수술 물을 다시 순환 담요 이상 stereotaxic 프레임에 마우스를 놓습니다. 단단히 귀에 바와 함께 머리를 확보.
  4. 밖으로 건조에서 동물의 눈에을 방지하기 위해, 눈 연고를 적용합니다.
  5. Dexamethasone (0.2 밀리그램 / 킬로그램)와 Carprofen가 (5 밀리그램 / 킬로그램) 각각의 뇌 및 / 또는 염증 반응의 붓기 방지하기 위해, subcutaneously, 관리할 수 있습니다.
  6. 수술을 시작하기 전에 70 % 알코올 및 betadine 세 교대 swipes와 함께 피부를 닦는하여 운영 지역을 소독.
  7. 모든 수술기구는 유리 비드 살균기를 사용하여 사전에 소독하고 있습니다. 에탄올로 소독되었습니다 가위를 사용하여 살다가 두 주동이의 방향으로 상처 거의 눈꺼풀에 도달, 두 경사 상처 뒤에있는 모든 머리의 기본을 따라 수평 절단, 시작, 두개골의 맨 위에 피부를 제거 그것은 정중선에서 수렴.
  8. lidocaine + 에피네프린 솔루션의 한 방울이 과도한 출혈이나 통증을 피하기 위해 골막에이 시점에서 적용됩니다. 메스로 두개골의 가장자리에 골막을 철회. 또한, 가볍게 목 뒤쪽의 근육을 철회.
  9. 부드럽게 마른 표면을 만들기 위해 메스로 두개골의 전체 노출 영역을 다쳤어요. 나중에 적용할 때 접착제가 잘 준수 수 있듯이 매우 중요합니다.
  10. 이미징 사이트가 선택되면, 하나는 두개골 창을 만들 준비가되어 있습니다. 첫째, 부드럽게 공압 치과 드릴 직경 약 4 mm의 원형 "을 그려."
  11. 약간 드릴링 후, 두개골 표면에 다시 lidocaine + 에피네프린 솔루션을 적용할 수 있습니다. 뼈의 매우 얇은 층이 남아있는 책임이 중지합니다. 이 방법을 제공하는 방법 느낄 수 craniotomy의 중앙에 부드럽게 밀어 하나는 일반적으로이 단계에 도달 알고 있습니다.
  12. 뼈 해면 구조 - - 뼈 trabeculae의 생리와 복용의 이점을 한 방울에 따라 매우 얇은 팁 포셉와 두개골에서 craniotomy 멀리 리프트. 그것은 두개골을 들어올려하고 두라의 출혈을 방지하므로 생리가 중요합니다.
  13. 이전에 두개골이 제거되었을 때 가끔 발생하는 작은 출혈을 멈추게하는 두라 메이터에 호수에 배어있다 Gelfoam을 적용합니다.
  14. 두라 메이터 표면을 건조되며 출혈이 없다는 것을 확인한 후, 부드럽게 두라 메이터 위에 멸균 5mm 유리 커버 슬립을 믿는다. (참고 : 다른 그룹도 두라 이상 낮은 용융 점 아가로 오스 (1.2 %)의 드롭을 배치하고 한천 위에 coverslip을 넣어.)
  15. 두개골에 반대 반구에 cyanocrylate 기반 접착제 한 방울을 적용합니다. 조심하는 것이 유리 밑에 넣고되지 동안 바늘의 도움으로, 부드럽게 모든 창 주변의 접착제를 적용합니다. 접착제 이제 두개골의 전체 표면 위에 얇은 레이어에 적용할 수 있습니다.
  16. 접착제가 건조되면, 그것을 보장하기 위해서, 또한 커버 슬립의 작은 림을 포함, 두개골 표면에 걸쳐 치과 아크릴을 혼합하고 그것을 적용해야합니다.
  17. 커버 슬립을 확보 후, 치과 아크릴과 창문 주위에 작은 우물을합니다. 또한, 치과 아크릴에 티타늄 막대를 삽입할 수 있습니다. 이 막대는 나중에 영상을위한 현미경의 무대에 안전하게 마우스를 첨부하는 데 사용됩니다. 그것은 두개골 창은 병렬가되도록, 바가 수준 있는지 확인하는 것이 중요합니다. 막대 아래에 종이를 게재하면 아크릴은 견고하면서 막대가 수준 유지하도록 할 수 있습니다.
  18. 치과 아크릴은 10 분 (강화) 치료 허용, 어떤 시점에 티타늄 막대 제자리에 고정합니다. 그것이 복구까지 따뜻한 새장에 동물을 배치합니다.
  19. 마취에서 회복 후, 동물은 당일 몇 군데하실 수 있습니다.

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Discussion

우리가 비디오에서 두 광자 현미경의 사용과 결합하여 보조 수치, 두개골 윈도우 준비에 나타난 것처럼, 생체내 네크로 텍스의 구조와 기능에서 공부하는 매우 강력한 도구입니다. 기술 관련 해부학 및 미세 수술 절차 및이 준비가 필요 기술에 익숙해지로 엄격한 훈련이 필요합니다. 단지 깨끗한 수술은 만성 이미징 사용할 수 있습니다. 두라이 과도하거나 구멍을 조작하면, 준비는 이미지에 사용해서는 안됩니다.

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Materials

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

DOWNLOAD MATERIALS LIST

References

  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).

Comments

71 Comments

  1. 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

    Reply
    Posted by: Anonymous
    March 25, 2008 - 8:05 AM
  2. 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 

    Reply
    Posted by: Anonymous
    March 25, 2008 - 1:49 PM
  3. 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

    Reply
    Posted by: Anonymous
    February 11, 2009 - 8:10 PM
  4. 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
     

    Reply
    Posted by: Anonymous
    April 17, 2009 - 1:38 PM
  5. Hi, Could you provide me with the manufacturer and model number of the pneumatic drill that you use?   Thanks a lot,   Dan

    Reply
    Posted by: Anonymous
    June 17, 2008 - 8:06 PM
  6. 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.      

    Reply
    Posted by: Anonymous
    June 17, 2008 - 9:48 PM
  7. Hi, Could you please tell me the size of the drill bit that you use during craniotomy? Thank you
    Paul

    Reply
    Posted by: paul z.
    October 21, 2014 - 4:31 PM
  8. Hello, Could you please tell me the dimensions of your titanium bar, and also its weight? Thanks for your help. James T. Russell

    Reply
    Posted by: Anonymous
    July 3, 2008 - 1:19 PM
  9. 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

    Reply
    Posted by: Anonymous
    July 7, 2008 - 2:20 PM
  10. 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

    Reply
    Posted by: Anonymous
    July 9, 2008 - 6:13 AM
  11. 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

    Reply
    Posted by: Anonymous
    July 9, 2008 - 5:35 PM
  12. 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

    Reply
    Posted by: Anonymous
    July 11, 2008 - 3:30 PM
  13. 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    

    Reply
    Posted by: Anonymous
    September 17, 2008 - 7:20 AM
  14. 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

    Reply
    Posted by: Anonymous
    September 17, 2008 - 1:36 PM
  15. Hi Ricardo and Ralph, We use a UV acrylic from Pentron Dental that sets in 10 seconds under UV light. Tom

    Reply
    Posted by: Anonymous
    November 1, 2008 - 11:17 PM
  16. 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

    Reply
    Posted by: Anonymous
    September 18, 2008 - 4:23 AM
  17. 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.  

    Reply
    Posted by: Anonymous
    September 18, 2008 - 3:26 PM
  18. 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.

    Reply
    Posted by: Anonymous
    March 18, 2009 - 1:15 PM
  19. Hi very nice video! Raquel Revilla-Sanchez Tufts University

    Reply
    Posted by: Anonymous
    September 27, 2008 - 7:11 PM
  20. 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

    Reply
    Posted by: Anonymous
    October 26, 2008 - 11:57 PM
  21. 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

    Reply
    Posted by: Anonymous
    October 27, 2008 - 1:28 AM
  22. 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

    Reply
    Posted by: Anonymous
    February 9, 2009 - 1:08 PM
  23. 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

    Reply
    Posted by: Anonymous
    February 10, 2009 - 3:05 PM
  24. 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 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

    Reply
    Posted by: Anonymous
    February 20, 2009 - 11:32 AM
  25. 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  

    Reply
    Posted by: Anonymous
    February 24, 2009 - 1:52 PM
  26. 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

    Reply
    Posted by: Anonymous
    February 20, 2009 - 5:14 PM
  27. 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

    Reply
    Posted by: Anonymous
    February 24, 2009 - 2:02 PM
  28. 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.

    Reply
    Posted by: Anonymous
    February 25, 2009 - 8:12 AM
  29. 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.

    Reply
    Posted by: Anonymous
    March 18, 2009 - 1:10 PM
  30. 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. 

    Reply
    Posted by: Anonymous
    March 9, 2009 - 9:50 AM
  31. 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

    Reply
    Posted by: Anonymous
    April 16, 2009 - 4:42 PM
  32. 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

    Reply
    Posted by: Anonymous
    April 17, 2009 - 11:44 AM
  33. 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

    Reply
    Posted by: Anonymous
    April 28, 2009 - 12:04 AM
  34. 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

    Reply
    Posted by: Anonymous
    May 6, 2009 - 3:27 AM
  35. Hi Ricardo
    Thank you very much for your share.Could you tell me where can I get the titanium bar?
    Thank you.
    Fei Li

    Reply
    Posted by: Anonymous
    August 25, 2009 - 7:07 AM
  36. Hello Fei,

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

    Ricardo

    Reply
    Posted by: Anonymous
    August 25, 2009 - 1:44 PM
  37. Hello
    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

    Reply
    Posted by: Anonymous
    August 31, 2009 - 3:38 AM
  38. 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.
    Ricardo

    Reply
    Posted by: Anonymous
    September 10, 2009 - 2:15 AM
  39. very good

    Reply
    Posted by: john d.
    November 6, 2009 - 12:38 PM
  40. 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!

    Reply
    Posted by: V C.
    May 14, 2010 - 12:06 PM
  41. 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.
    Ricardo

    Reply
    Posted by: Anonymous
    June 1, 2010 - 6:20 PM
  42. Hi, would you please send me information on the heated blanket you are using during surgery? Many thanks, Ute

    Reply
    Posted by: Ute F.
    August 13, 2010 - 10:44 AM
  43. Hello Ute
    We use a water circulating pump Gaymar T/Pump TP-500. It's sold from many different sources.
    Hope this helps

    Reply
    Posted by: Anonymous
    August 13, 2010 - 2:49 PM
  44. 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

    Reply
    Posted by: Ute F.
    August 16, 2010 - 12:10 PM
  45. We bought the standard ones and fold them, although there are smaller sizes. We ordered directly from Gaymar

    Reply
    Posted by: Anonymous
    August 17, 2010 - 1:18 PM
  46. Thank you, Ricardo! Ute

    Reply
    Posted by: Ute F.
    August 17, 2010 - 1:56 PM
  47. 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.
    Thanks.

    Reply
    Posted by: Anonymous
    May 27, 2011 - 11:24 AM
  48. The bar has a tapped hole so you can use a screw to attach the bar to a holder

    Ricardo

    Reply
    Posted by: Anonymous
    May 27, 2011 - 2:45 PM
  49. The titanium bar has a tapped hole so you can use a screw to attach the bar to a holder

    Ricardo

    Reply
    Posted by: Anonymous
    May 27, 2011 - 2:45 PM
  50. Ok, just another thing, what kind of holder you use? You developed the holder? or, could you provide me a serial number? Thanks

    Reply
    Posted by: Anonymous
    May 31, 2011 - 9:49 AM
  51. The holder is custom made

    Ricardo

    Reply
    Posted by: Anonymous
    May 31, 2011 - 2:13 PM
  52. 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?

    Reply
    Posted by: Anonymous
    June 13, 2011 - 5:00 PM
  53. I don't know. JoVE staff came with their equipment and filmed the video.

    Reply
    Posted by: Anonymous
    June 14, 2011 - 3:23 PM
  54. 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?
    Thank's
    Rafael

    Reply
    Posted by: Anonymous
    September 22, 2011 - 5:24 PM
  55. Thanks.
    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!
    Carlos

    Reply
    Posted by: Anonymous
    September 22, 2011 - 5:32 PM
  56. 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!

    Reply
    Posted by: Anonymous
    October 11, 2011 - 9:45 PM
  57. 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
    Ricardo

    Reply
    Posted by: Anonymous
    October 12, 2011 - 2:35 PM
  58. 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 !

    Reply
    Posted by: Anonymous
    October 12, 2011 - 9:33 PM
  59. 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.

    Reply
    Posted by: Anonymous
    October 14, 2011 - 2:35 PM
  60. 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!

    Reply
    Posted by: Anonymous
    October 26, 2011 - 11:21 PM
  61. We use this: Ortho-Jet Acrylic Powder clear (1330CLR) + Ortho-Jet Acrylic Liquid clear (1303CLR) from Lang Dental

    Reply
    Posted by: Anonymous
    October 28, 2011 - 5:57 PM
  62. We use this: Ortho-Jet Acrylic Powder clear (1330CLR) + Ortho-Jet Acrylic Liquid clear (1303CLR) from Lang Dental

    Reply
    Posted by: Anonymous
    October 28, 2011 - 5:56 PM
  63. 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.

    Reply
    Posted by: Anonymous
    November 21, 2011 - 4:05 AM
  64. 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.
    Ricardo

    Reply
    Posted by: Anonymous
    January 23, 2012 - 8:30 PM
  65. 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.

    Reply
    Posted by: Anonymous
    January 23, 2012 - 12:56 PM
  66. 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
    Ricardo

    Reply
    Posted by: Anonymous
    January 23, 2012 - 8:50 PM
  67. 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!

    Reply
    Posted by: Anonymous
    April 8, 2012 - 7:51 PM
  68. Hello,
    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.
    Ricardo

    Reply
    Posted by: Anonymous
    April 9, 2012 - 3:31 PM
  69. Hello Ricardo,

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

    Reply
    Posted by: Anonymous
    April 9, 2012 - 3:48 PM
  70. 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.

    Reply
    Posted by: Anonymous
    April 9, 2012 - 4:39 PM
  71. 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.
    Thanks!
    David

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

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