欢迎您, 登录 | 注册

首页 | English

X
加载中

Stereotaxic injection is an invaluable tool for the creation of site-targeted lesions, injection of anatomical tracers, gene delivery by recombinant adeno-associated viruses and lentiviruses in mice and rats. Stereotaxic injection of LPS or 6-hydroxydopamine has been used to establish animal models of Parkinson’s disease, the most common neurodegenerative movement disorder. This protocol allows the investigation of central nervous system development and disease mechanisms. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

Thanks for your further question/comment. It has been sent to the author(s) of this protocol. You will receive a notification once your question/comment is addressed again by the author(s).
Meanwhile, it would be great if you could help us to spread the word about Bio-protocol.

X

[Bio101] Stereotaxic Injection of LPS or Your Reagents of Choice into Rat Substantia Nigra
[Bio101] 向大鼠脑黑质立体定向注射LPS或其他测试试剂

神经科学 > 神经系统疾病
作者: Huiming Gao
Huiming GaoAffiliation: Neuropharmacology Section, National Institute of Environmental Health Sciences, National Institutes of Health, Research Triangle Park, NC, USA
For correspondence: gao2@niehs.nih.gov
Bio-protocol author page: a17
11/20/2011, 5328 views, 0 Q&A
DOI: https://doi.org/10.21769/BioProtoc.154

[Abstract] Stereotaxic injection is an invaluable tool for the creation of site-targeted lesions, injection of anatomical tracers, gene delivery by recombinant adeno-associated viruses and lentiviruses in mice and rats. Stereotaxic injection of LPS or 6-hydroxydopamine has been used to establish animal models of Parkinson’s disease, the most common neurodegenerative movement disorder. This protocol allows the investigation of central nervous system development and disease mechanisms. This protocol has been developed and improved over the years by various researchers in Dr. Hong’s lab, especially Dr. Bin Liu.

[Abstract] 立体定向注射是一种极为宝贵的工具,可在小鼠和大鼠中创建定点靶向病灶、解剖示踪剂注射、重组腺相关病毒与慢病毒载体的基因传递。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。此实验方法可供中枢神经系统发育及发病机制的调查。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. Two-month-old male F344 rats, body weight 220-250 g
  2. Nembutal
  3. Carprofen
  4. Betadine
  5. 70% ethanol
  6. Phosphate buffered saline (PBS)
  7. 4% paraformaldehyde
  8. Ocular lubricant (Puralube)
  9. LPS (Escherichia coli 0111: B4) (Sigma-Aldrich)
  10. Sterile normal saline (0.9%) or other vehicle for your reagents
  11. LPS stock solution (see Recipes)

Equipment

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

Procedure

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

  2. Analgesic
    Carprofen, 5 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. 4.8 mm posterior to the bregma.
    2. 1.7 mm lateral to the midline.
    3. 8.2 mm ventral to the surface of skull (Paxinos and Watson, 1986).
      Note: Differences in rat strains and age might require an adjustment to the coordinates for the injection.

  5. Surgical procedure
    1. Stabilize the head of the rat 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, the rat is 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.

References

  1. Liu, B., Jiang, J. W., Wilson, B. C., Du, L., Yang, S. N., Wang, J. Y., Wu, G. C., Cao, X. D. and Hong, J. S. (2000). Systemic infusion of naloxone reduces degeneration of rat substantia nigral dopaminergic neurons induced by intranigral injection of lipopolysaccharide. J Pharmacol Exp Ther 295(1): 125-132.
  2. Paxinos, G. and Watson, C. (1986). The Rat Brain in Stereotaxic Coordinates, 2nd edn. Orlando, FL: Academic Press.

材料与试剂

 

1.        两月龄雄性F344大鼠,体重220-250g

2.        戊巴比妥Nembutal 

3.        卡洛芬(Carprofen

4.        LPS (大肠杆菌Escherichia coli 0111:B4; Sigma)

5.        无菌生理盐水 (0.9%)或其他所选试剂的媒介物

6.        眼部润滑剂(Puralube)

 

设备

 

1.       机动微量注射泵

2.       小型动物立体定位仪(大鼠立体定位仪)

3.       微量注射器具

4.       牙钻和 #1小牙钻

5.       切片刀

6.       解剖刀 (#10)

7.       组织

8.       纱布

9.       Autoclips小手术夹/缝合材料

 

实验步骤

 

1.       动物麻醉:戊巴比妥50 mg / kg腹腔注射

2.       镇痛:卡洛芬,5毫克/公斤手术时间皮下注射。

3.       动物制备:

1)      夹起头顶的毛Clip hair from the top of the head.

2)      依次用优碘与70%乙醇消毒皮肤。

3)      进行麻醉施用眼部润滑剂(Puralube),以防止眼睛干燥

4.       用于注射的坐标:

4)      前囟门后 4.8 mm

5)      外侧向中线1.7 mm

6)      头骨表面腹侧8.2 mm

(Paxinos and Watson 1986)

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。 这是耳栏正确定位头部关键。这可通过从左到右移动鼻子,对面的眼睛会斜视进行验证。

注意:没必要去正确定位穿刺耳膜

2)      在头皮中线切一个 5 mm 切口。

3)      为防出血,用解剖刀柄的钝边轻轻刮除紧接骨头的骨膜结缔组织

4)      确定颅缝线,前囟点 和人字点 在顶颅骨内板用小牙钻一个洞(大鼠脑立体图集确定坐标)

5)      孔将穿透完整的头骨,但不是硬脑膜。硬脑膜是非常坚硬的膜,但可以很容易地用锋利的皮下注射针头切片。

6)      一个连接到显微注射仪器的预先加载30ga口径的微量注射器通过颅骨孔洞预测深度。注射将在2min内进行,由机动微量注射泵控制

7)      重复步骤4至第6步注射2μl无菌生理盐水(0.9%)到对侧大脑。

8)      注射后,针尖固定2 min

9)      从大脑慢慢移出针头。

10)  Autoclips小手术夹或丝缝线关闭皮肤切口

6.       术后护理:

1)      监视动物指导从麻醉状态恢复

2)      每天监视切口是否有任何分泌物、红肿或开裂现象.

3)      如果动物出现运动不节省,不活动或不愿动弹,立即兽医科联系。

4)      10-14天后移去Autoclips小手术夹或拆线。

5)      在所需要的时间点,大鼠将被麻醉并用PBS进行心脏灌流,然后进行PBS缓冲4%多聚甲醛进行免疫组织化学。

 

配方

 

1.       LPS 用无菌生理盐水(0.9%)准备5 mg/ml储液,并用小分装在4°C保存

 

参考文献

 

1.         Liu B., Jiang J.W., Wilson B.C., Du L., Yang S.N., Wang J.Y., Wu G.C., Cao X.D., Hong J.S. (2000). Systemic infusion of naloxone reduces degeneration of rat substantia nigral dopaminergic neurons induced by intranigral injection of lipopolysaccharide. Journal of Pharmacology and Experimental Therapeutics 295(1): 125-32. 

2.         Paxinos G., Watson C. (1986) The Rat Brain in stereotaxic coordinates, 2nd edn. Orlando, FL: Academic Press.

 

English
中文翻译

免责声明

为了向广大用户提供经翻译的内容,www.bio-protocol.org 采用人工翻译与计算机翻译结合的技术翻译了本文章。基于计算机的翻译质量再高,也不及 100% 的人工翻译的质量。为此,我们始终建议用户参考原始英文版本。 Bio-protocol., LLC对翻译版本的准确性不承担任何责任。

X


How to cite this protocol: Gao, H. (2011). Stereotaxic Injection of LPS or Your Reagents of Choice into Rat Substantia Nigra. Bio-protocol Bio101: e154. DOI: 10.21769/BioProtoc.154; Full Text



可重复性反馈:

  • 添加图片
  • 添加视频

我们的目标是让重复别人的实验变得更轻松,如果您已经使用过本实验方案,欢迎您做出评价。我们鼓励上传实验图片或视频与小伙伴们(同行)分享您的实验心得和经验。(评论前请登录)

问题&解答:

  • 添加图片
  • 添加视频

(提问前,请先登陆)bio-protocol作为媒介平台,会将您的问题转发给作者,并将作者的回复发送至您的邮箱(在bio-protocol注册时所用的邮箱)。为了作者与用户间沟通流畅(作者能准确理解您所遇到的问题并给与正确的建议),我们鼓励用户用图片或者视频的形式来说明遇到的问题。由于本平台用Youtube储存、播放视频,作者需要google 账户来上传视频。


登陆 | 注册
引用格式
分享
Twitter Twitter
LinkedIn LinkedIn
Google+ Google+
Facebook Facebook