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Stereotaxic Infusion of LPS into Rat Substantia Nigra via Osmotic Minipump
通过微型渗透泵向大鼠脑黑质立体定向注射LPS   

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Abstract

Stereotaxic infusion of LPS or your reagent of choice is an invaluable tool for the creation of chronic site-targeted lesions. Stereotaxic infusion of LPS has been used to establish chronic animal model of Parkinson’s disease, the most common neurodegenerative movement disorder. This protocol is especially useful to established chronic disease models and to study mechanisms of chronic central nervous system diseases.

Materials and Reagents

  1. Eight-week-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. 0.9% sterile normal saline or other vehicle for your reagents
  11. LPS stock solution (see Recipes)

Equipment

  1. Small-animal stereotaxic apparatus (rat stereotaxic apparatus)
  2. Osmotic minipump (Alza Corporation)
  3. Dental drill and #1 burrs
  4. Dental cement
  5. Stereotaxic frame
  6. Cannula (stainless steel with cap)
  7. Microknife
  8. Scalpel (#10)
  9. Tissue forceps
  10. Gauze
  11. 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 infusion
    1. 4.8 mm posterior to the bregma.
    2. 1.7 mm lateral to the midline.
    3. 8.0 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 10 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. The cannula is lowered into the hole to a depth previously determined by stereotaxic atlas.
    7. To secure the cannula to the skull, mix dental cement and slowly build a base, making sure that the side is smooth next to the cut scalp.
    8. Remove cement from any adhering skin.
    9. Implant an Alzet osmotic minipump under the skin on the back of the animal.
    10. A specialized cannula (30 gauge, Plastics One) with a side port, which allows for attachment of a polyethylene tube, is connected to the Alzet minipump.
    11. The tube is tunneled to the back of the animal where the minipump is implanted subcutaneously.
    12. Close the skin incision with autoclips or silk suture.

  6. Post-operative care
    1. Monitor animal until recovered from anesthesia.
    2. Administer Carprofen (5 mg/kg subcutaneous injection) for post-operation pain upon recovery from anesthesia.
    3. Monitor incision daily for any discharge, swelling or dehiscence.
    4. If animal appears unthrifty, inactive or reluctant to move, contact the Veterinary Medicine Section immediately.
    5. Authclip/suture removal in 10-14 days.
    6. 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., 2002; Paxinos and Watson, 1986).

References

  1. Gao, H. M., Jiang, J., Wilson, B., Zhang, W., Hong, J. S. and Liu, B. (2002). Microglial activation-mediated delayed and progressive degeneration of rat nigral dopaminergic neurons: relevance to Parkinson's disease. J Neurochem 81(6): 1285-1297.
  2. Paxinos, G. and Watson, C. (1986). The Rat Brain in Stereotaxic Coordinates, 2nd edn. Orlando, FL: Academic Press.

简介

立体定位输注LPS或您选择的试剂是创建慢性位点靶向病变的非常有用的工具。 LPS的立体定位输注已经用于建立帕金森病的慢性动物模型,帕金森病是最常见的神经变性运动障碍。 这个协议对建立慢性疾病模型和研究慢性中枢神经系统疾病的机制特别有用。

材料和试剂

  1. 八周龄雄性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. 0.9%无菌生理盐水或其他用于您的试剂的载体
  11. LPS储备溶液(见配方)

设备

  1. 小动物立体定位装置(大鼠立体定位装置)
  2. 渗透微型泵(Alza Corporation)
  3. 牙钻和#1毛刺
  4. 牙科用水泥
  5. 立体框架
  6. 插管(不锈钢带帽)
  7. Microknife
  8. 手术刀(#10)
  9. 组织钳
  10. 纱布
  11. 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,5mg/kg在手术时给予皮下注射
  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. 管理镇痛药。
    4. 使用眼部润滑剂以防止眼睛干燥。

  4. 用于输注的坐标
    1. 前囱后方4.8 mm。
    2. 在中线的侧向1.7mm。
    3. 8.0 mm至颅骨表面(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. 通过使用耳杆稳定立体定位框架中的老鼠的头。 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10mm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,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. 套管下降到孔中达到由立体定位图谱预先确定的深度。
    7. 为了将套管固定到颅骨,混合牙科骨水泥,慢慢地建立一个基地,确保侧面在切割的头皮旁边光滑。
    8. 从任何粘附的皮肤上除去水泥。
    9. 种植一个Alzet渗透微型泵在动物的背部皮肤下。
    10. 具有允许连接聚乙烯管的侧端口的专用插管(30规格,Plastics One)连接到Alzet微型泵。
    11. 管被隧道传送到动物的背部,其中微型泵皮下植入。
    12. 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. 在麻醉恢复后施用卡洛芬(5mg/kg皮下注射)用于术后疼痛。
    3. 每天监测切口的放电,肿胀或开裂
    4. 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。
    5. 在10-14天内摘除/缝合缝合。
    6. 在所需的时间点,大鼠将被麻醉并经心脏灌注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的实验室的各种研究者,特别是Liu Bin博士多年来发展和改进(高等人,2002; Paxinos和Watson,1986)。

参考文献

  1. Gao,H.M.,Jiang,J.,Wilson,B.,Zhang,W.,Hong,J.S.and Liu,B。(2002)。 小胶质细胞活化介导的大鼠黑质多巴胺能神经元的延迟和进行性变性:与帕金森病的相关性。 a> J Neurochem 81(6):1285-1297
  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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Copyright: © 2012 The Authors; exclusive licensee Bio-protocol LLC.
引用:Gao, H. (2012). Stereotaxic Infusion of LPS into Rat Substantia Nigra via Osmotic Minipump. Bio-protocol 2(8): e152. DOI: 10.21769/BioProtoc.152.
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