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Stereotaxic injection is an attractive approach for studying genetic, cellular and circuit functions in the brain. Injection of anatomical tracers, site-targeted lesions and gene delivery by recombinant adeno-associated viruses and lentiviruses in mice are powerful tools to study nervous system development and disease mechanisms. Stereotaxic injection of LPS or 6-hydroxydopamine has been used to establish animal models of Parkinson’s disease, the most common neurodegenerative movement disorder. Importantly, this protocol allows the manipulation of gene expression in the targeted rodent brain regions and even targeted cell types or a subpopulation of cells in the injected region at any postnatal developmental stage up to adulthood.

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Stereotaxic Injection of LPS into Mouse Substantia Nigra
向小鼠脑黑质立体定向注射LPS

神经科学 > 神经系统疾病
作者: Huiming Gao
Huiming GaoAffiliation: National Institute of Environmental Health Sciences, Research Triangle Park, NC, USA
For correspondence: gao2@niehs.nih.gov
Bio-protocol author page: a17
Vol 2, Iss 8, 4/20/2012, 11879 views, 4 Q&A
DOI: https://doi.org/10.21769/BioProtoc.153

[Abstract] Stereotaxic injection is an attractive approach for studying genetic, cellular and circuit functions in the brain. Injection of anatomical tracers, site-targeted lesions and gene delivery by recombinant adeno-associated viruses and lentiviruses in mice are powerful tools to study nervous system development and disease mechanisms. Stereotaxic injection of LPS or 6-hydroxydopamine has been used to establish animal models of Parkinson’s disease, the most common neurodegenerative movement disorder. Importantly, this protocol allows the manipulation of gene expression in the targeted rodent brain regions and even targeted cell types or a subpopulation of cells in the injected region at any postnatal developmental stage up to adulthood.

[Abstract] 立体定向注射是一种研究大脑基因、细胞、线路回路功能的遗传吸引人的方法。解剖示踪剂注射、创建定点靶向病灶、重组腺相关病毒与慢病毒载体的基因传递是研究神经发育和疾病机制的强大工具。立体定向注射LPS或6-羟多巴胺已被用于建立帕金森病(Parkinson’s disease)这个最常见的神经退行性运动障碍疾病的动物模型。更重要的是,该方法可让基因表达操纵在针对性的啮齿类动物的大脑区域,甚至在定向的细胞类型或注射区域的一个细胞亚群进行,并且在后天发育的任何阶段直到成年进行。此实验方法经Dr. Hong实验室不同研究人员多年的发展和完善,尤其是Dr. Bin Liu.

Materials and Reagents

  1. Eight-week-old male C57 mice or a variety of transgenic/knockout mice, body weight 20-25 g
  2. Nembutal
  3. Buprenorphine
  4. LPS (Escherichia coli 0111: B4) (Sigma-Aldrich)
  5. Sterile normal saline (0.9%) or other vehicle for your reagents
  6. Ocular lubricant (Puralube)
  7. 70% ethanol
  8. 4% paraformaldehyde
  9. Betadine
  10. Phosphate buffered saline (PBS)
  11. LPS stock solution (see Recipes)

Equipment

  1. Motorized microinjection pump
  2. Small-animal stereotaxic apparatus (mouse stereotaxic apparatus)
  3. Microinjection apparatus
  4. Dental drill and #1 burrs
  5. Microknife
  6. Scalpel (#10)
  7. Tissue forceps
  8. Gauze
  9. Autoclips/suture materials
  10. Stereotaxic frame

Procedure

  1. Animal anesthesia
    Nembutal 50 mg/kg intraperitoneal injection.

  2. Analgesic
    Buprenorphine, 0.1 mg/kg subcutaneous injection given at the time of surgery.

  3. Animal preparation
    1. Clip hair from the top of the head.
    2. Decontaminate skin with betadine followed by 70% ethanol.
    3. Administer the analgesic.
    4. Apply an ocular lubricant to prevent drying of the eyes.

  4. The coordinates used for the injection
    1. 3.0 mm posterior to the bregma.
    2. 1.3 mm lateral to the midline.
    3. 4.7 mm ventral to the surface of the skull.
      Note: Differences in mouse age and body weight might require an adjustment to the coordinates for the injection.

  5. Surgical procedure
    1. Stabilize the head of the mouse in the stereotaxic frame by using the ear bars. It is critical for the head to be positioned correctly by the ear bars. This can be verified by moving the nose right to left and the eye on the opposite side will squint.
      Note: There is no need to puncture eardrums for proper positioning.
    2. Make a 5 mm incision in the midline of the scalp.
    3. To prevent bleeding, gently scrape away the periosteal connective tissue that adheres to the bone with the blunt edge of the scalpel handle.
    4. The cranial sutures, bregma and lambda will be identified and a hole will be drilled with a small dental drill in the parietal skull plate (coordinates to be determined by stereotaxic atlas of rat brain).
    5. The hole will penetrate the full skull but not the dura mater. The dura is a very tough membrane but can easily be sliced with a sharp hypodermic needle.
    6. A pre-loaded 30 g a microinjection syringe attached to the microinjection apparatus is slowly inserted into the brain to predetermined depth through the opening in the skull. The injection will be conducted over a period of 2 min and controlled by a motorized microinjection pump.
    7. Repeat step 4 to step 6 to injection 2 µl of sterile normal saline (0.9%) into the opposite side of the brain.
    8. After the injection, the needle will be kept in place for 2 min.
    9. Remove the needle slowly out of the brain.
    10. Close the skin incision with autoclips or silk suture.

  6. Post-operative care
    1. Monitor animal until recovered from anesthesia.
    2. Monitor incision daily for any discharge, swelling or dehiscence.
    3. If animal appears unthrifty, inactive or reluctant to move, contact the Veterinary Medicine Section immediately.
    4. Authclip/suture removal in 10-14 days.
    5. At desired time points, rat will be anesthetized and transcardially perfused with PBS, followed by PBS-buffered 4% paraformaldehyde for Immunohistochemistry.

Recipes

  1. LPS prepared as a stock solution of 5 mg/ml in sterile normal saline (0.9%) and stored in small aliquots at 4 °C.

Acknowledgments

This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu (Gao et al., 2008).

References

  1. Gao, H. M., Kotzbauer, P. T., Uryu, K., Leight, S., Trojanowski, J. Q. and Lee, V. M. (2008). Neuroinflammation and oxidation/nitration of alpha-synuclein linked to dopaminergic neurodegeneration. J Neurosci 28(30): 7687-7698.

材料和试剂

  1. 8周龄雄性C57小鼠或各种转基因/敲除小鼠,体重20-25g
  2. Nembutal
  3. 丁丙诺啡
  4. LPS(大肠杆菌 0111:B4)(Sigma-Aldrich)
  5. 无菌生理盐水(0.9%)或其他试剂的载体
  6. 眼用润滑剂(Puralube)
  7. 70%乙醇
  8. 4%多聚甲醛
  9. betadine
  10. 磷酸盐缓冲盐水(PBS)
  11. LPS储备溶液(见配方)

设备

  1. 电动显微注射泵
  2. 小动物立体定位装置(小鼠立体定位装置)
  3. 显微注射装置
  4. 牙钻和#1毛刺
  5. Microknife
  6. 手术刀(#10)
  7. 组织钳
  8. 纱布
  9. 自动剪切/缝合材料
  10. 立体框架

程序

  1. 动物麻醉
    Nembutal 50mg/kg腹膜内注射
  2. 止痛剂
    在手术时给予丁丙诺啡,0.1mg/kg皮下注射
  3. 动物准备
    1. 从头顶部剪下头发。
    2. 用betadine,然后用70%乙醇净化皮肤。
    3. 管理止痛剂。
    4. 使用眼部润滑剂以防止眼睛干燥。

  4. 用于注射的坐标
    1. 前囟后3.0 mm。
    2. 距中线1.3毫米。
    3. 4.7毫米腹侧到颅骨表面 注意:小鼠年龄和体重的差异可能需要调整注射的坐标。

  5. 外科手术
    1. 通过使用耳杆稳定立体定位框架中的鼠标头。 关键在于头部由耳杆正确定位。 这可以通过从右向左移动鼻子来验证,而相反侧的眼睛会眯起 注意:没有必要穿刺鼓膜以进行正确的定位。
    2. 在头皮的中线做一个5毫米的切口。
    3. 为了防止出血,用手术刀手柄的钝边轻轻刮掉附着在骨上的骨膜结缔组织。
    4. 颅骨缝合线,前囟和λ将被识别,并且将在顶叶颅骨板中用小牙钻钻孔(坐标由大鼠脑的立体定位图谱确定)。
    5. 孔将穿透完整的头骨,但不是硬脑膜。硬膜是一个非常坚韧的膜,但可以很容易用锋利的皮下注射针切片
    6. 将预先加载的30g连接到显微注射装置的显微注射注射器通过颅骨中的开口缓慢地插入脑中达预定深度。注射将在2分钟的时间段内进行并由电动显微注射泵控制。
    7. 重复步骤4到步骤6注射2μl无菌生理盐水(0.9%)到大脑的另一侧。
    8. 注射后,针头将保持在原位2分钟。
    9. 慢慢地从大脑中取出针头。
    10. 用高压灭菌器或丝线缝合皮肤切口。

  6. 术后护理
    1. 监测动物,直到从麻醉中恢复。
    2. 每天监测切口的放电,肿胀或开裂
    3. 如果动物表现为不节俭,不活动或不愿意移动,请立即联系兽医科。
    4. 在10-14天内摘除/缝合缝合。
    5. 在所需的时间点,大鼠将被麻醉,经心脏灌注PBS,然后用PBS缓冲的4%多聚甲醛进行免疫组织化学。

食谱

  1. LPS制备为在无菌生理盐水(0.9%)中的5mg/ml储备溶液,并在4℃下以小等分试样储存。

致谢

这个协议已经由Hong博士实验室的各种研究者,特别是Liu Bin博士多年来开发和改进(Gao等人,2008)。

参考文献

  1. Gao,H.M.,Kotzbauer,P.T.,Uryu,K.,Leight,S.,Trojanowski,J.Q.and Lee,V.M。(2008)。 与多巴胺能神经变性相关的α-突触核蛋白的神经炎症和氧化/硝化。 J Neurosci 28(30):7687-7698。
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How to cite this protocol: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Gao, H. (2012). Stereotaxic Injection of LPS into Mouse Substantia Nigra. Bio-protocol 2(8): e153. DOI: 10.21769/BioProtoc.153; Full Text
  2. Gao, H. M., Kotzbauer, P. T., Uryu, K., Leight, S., Trojanowski, J. Q. and Lee, V. M. (2008). Neuroinflammation and oxidation/nitration of alpha-synuclein linked to dopaminergic neurodegeneration. J Neurosci 28(30): 7687-7698.




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    登陆 | 注册
    2/23/2015 8:48:58 AM  

    注:由于 大鼠 品系和年龄的差异,可能需要调整注射坐标。

    5. 外科手术步骤:

    1) 使用耳栏在立体框架中稳定 大鼠 头部。

    大鼠or小鼠

    Reply

    Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
    You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

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    5/29/2013 2:05:02 PM  

    Rohan Gandhi
    Vertex Pharmaceuticals

    Are there any detailed instructions for inserting the ear bars in the mouse ear canal? Also, do you have a photo of your experimental set-up?

    6/14/2013 12:52:03 AM  

    Xuecai Ge
    University of California, Merced

    You may check out the following reference:
    Geiger B.M., Frank L.E., Caldera-Siu A.D., Pothos E.N. (2008). Survivable Stereotaxic
    Surgery in Rodents . JoVE. 20. http://www.jove.com/index/Details.stp?ID=880, doi:
    10.3791/880

    Hope it helps.

    6/14/2013 12:52:47 AM  

    Xuecai Ge
    University of California, Merced

    You may check out the following reference:
    Geiger B.M., Frank L.E., Caldera-Siu A.D., Pothos E.N. (2008). Survivable Stereotaxic
    Surgery in Rodents . JoVE. 20. http://www.jove.com/index/Details.stp?ID=880, doi:
    10.3791/880

    Hope it helps.

    Reply

    Please login to post your questions/comments. Your questions will be directed to the authors of the protocol. The authors will be requested to answer your questions at their earliest convenience. Once your questions are answered, you will be informed using the email address that you register with bio-protocol.
    You are highly recommended to post your data (images or even videos) for the troubleshooting. For uploading videos, you may need a Google account because Bio-protocol uses YouTube to host videos.

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    1/26/2013 2:25:21 PM  

    A picture of mouse skull with the coordinates
    would be very helpful.

    1/29/2013 11:54:05 PM  

    Xuecai Ge
    University of California, Merced

    I don't have such picture in hand. But online you can find a handful of them. Please check it out in the following two references:

    stereotaxic inj-1: from "Retrovirus-mediated single-cell gene knockout technique in adult newborn neurons in vivo". Tashiro,Zhao&Gage, Nature Protocols 1, 3049 - 3055 (2007).

    Stereotaxic inj-2: http://play.psych.mun.ca/~smilway/surgery-frame.html . The picture will show up by clicking "position the tip of the drill directly over Bregma."

    Reply

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    8/2/2012 6:02:38 PM  

    Which motorized microinjection pump did you use. Thanks, Claudia

    8/7/2012 11:24:28 PM  

    Huiming Gao (Author)
    National Institute of Environmental Health Sciences, Research Triangle Park, USA

    I used the KDS310 (Nano Pump) from KD Scientific Inc., which is used exclusively with micro syringes.

    Reply

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