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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.

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[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, 5821 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.

材料和试剂

  1. 两个月大的雄性F344大鼠,体重220-250克
  2. Nembutal
  3. Carprofen
  4. betadine
  5. 70%乙醇
  6. 磷酸盐缓冲盐水(PBS)
  7. 4%多聚甲醛
  8. 眼用润滑剂(Puralube)
  9. LPS(大肠杆菌 0111:B4)(Sigma-Aldrich)
  10. 无菌生理盐水(0.9%)或其他试剂的载体
  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(大鼠立体定位装置)
  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. 立体框架
  10. 自动剪切/缝合材料

程序

  1. 动物麻醉
    Nembutal 50mg/kg腹膜内注射
  2. 止痛剂
    卡洛芬,在手术时给予5mg/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. 用于注入的坐标
    1. 前囱后方4.8 mm。
    2. 距中线1.7毫米。
    3. 鼻间隔至颅骨表面8.2 mm(Paxinos和Watson,1986) 注意:大鼠品系和年龄的差异可能需要调整注射的坐标。

  5. 外科手术
    1. 通过使用耳朵稳定立体定位框架中的大鼠的头部   酒吧。 头部由耳朵正确定位是至关重要的   酒吧。 这可以通过从右向左移动鼻子和眼睛来验证   在对面会眯起。
      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,轻轻刮掉骨膜结缔组织 组织粘附到具有手术刀的钝边的骨 句柄。
    4. 头颅缝合线,前囟和λ将被识别   并在顶部钻一个小牙钻 颅骨板(坐标由大鼠的立体定位图谱确定 脑)。
    5. 孔将穿透完整的头骨,但不是硬脑膜 母亲。 硬膜是一个非常坚韧的膜,但可以很容易地用一个切片   尖锐的皮下注射针
    6. 预加载30 g显微注射 将连接到显微注射装置的注射器缓慢插入   大脑通过颅骨开口的预定深度。 的 注射将在2分钟的时间内进行并由a控制 电动显微注射泵。
    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,将大鼠麻醉并经心脏 用PBS灌注,然后用PBS缓冲的4%多聚甲醛 免疫组化。

食谱

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

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.and Hong,J.S。(2000)。 全身输注纳洛酮 减少由脑内注射脂多糖诱导的大鼠黑质多巴胺能神经元的变性。 Pharmacol Exp Ther 295(1):125-132。
  2. Paxinos,G.and Watson,C。(1986)。 大鼠脑立体定位坐标,第二版。 Orlando,FL:Academic Press
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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



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