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Peripheral Nerve Injury: a Mouse Model of Neuropathic Pain
外周神经损伤:神经性疼痛小鼠模型   

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Abstract

Neuropathic pain is one of the highly debilitating chronic pain conditions, for which, currently, there is no therapeutic treatment. In order to reveal the underlying mechanism for neuropathic pain, various animal models have been established (Burma et al., 2016). This protocol describes how to prepare spinal nerve injury model (Kim and Chung, 1992; Rigaud et al., 2008; Masuda et al., 2016), one of the most frequently-used and highly reproducible models in which multiple alterations occur both in the peripheral and central nervous system.

Keywords: Neuropathic pain(神经性疼痛), Nerve injury(神经损伤), Spinal cord(脊髓), Mouse(小鼠), Dorsal root ganglion (DRG)(背根神经节(DRG)), Allodynia(触诱发痛)

Background

Revealing the underlying mechanism of neuropathic pain is necessary to develop effective therapy for its optimal management. Therefore, various animal models for neuropathic pain have been developed. In particularly, rodent models have been frequently used because they are highly reproducible and exhibit pain hypersensitivity that is also observed in patients with neuropathic pain. In this protocol, we describe how to prepare the mouse spinal nerve injury model.

Materials and Reagents

  1. 5-0 silk suture (Alfresa Pharma, catalog number: GA05SW )
  2. Sterile scalpel blades (FATHER Safety Razor, catalog number: No.10 )
  3. Wild-type C57BL/6 mice (6-15 weeks old) (Japan Clea)
  4. Isoflurane (Mylan)
  5. 75 % ethanol (Takasugi Pharmaceutical)

Equipment

  1. Electric clippers (Daito Electric Machine Industry, catalog number: 605AD , Special C)
    Note: This product has been discontinued.
  2. Agricola retractor (Fine Science Tools, catalog number: 17005-04 )
  3. Forceps (NATSUME SEISAKUSHO, catalog number: A-14 )
  4. Vannas spring scissors (Fine Science Tools, catalog number: 15070-08 )
  5. Electric drill (URAWA Kogyo, model: MINITORJET UC210 )
  6. Scalpel holder (NATSUME SEISAKUSHO, catalog number: D-11 )
  7. Heating pad (VIVARIA, catalog number: MP-916-NV)
  8. Double sided micro spatula (Fine Science Tools, catalog number: 10091-12 )
  9. Isoflurane dispenser (FORWICK, MURACO Medical)

Procedure

Ethic Local animal ethics regarding animal housing and animal experiment need to be followed.

  1. In order to induce anesthesia, put the mouse in the plastic chamber where 5% isoflurane is provided through a tube to the dispenser (Figure 1). It takes approximately 30 sec.


    Figure 1. Inhalational anesthesia with isoflurane. The mouse was anesthetized via an inhalation mask using 2-3% isoflurane.

  2. Anesthetize the mouse with 2-3% inhaled isoflurane by setting on the board with the head placed into a mask connected to the dispenser (Figure 1).
  3. Make sure that the mouse is adequately anesthetized by applying pressure on the mouse toe with forceps (toe pinch).
  4. Remove hair from the proper region of the back of the mouse with electric clippers, and make a small incision with a fine scalpel from the lumbar L3 to the sacral S1 vertebra after cleaning with 75% ethanol (Figure 2).


    Figure 2. Small incision on the back skin. A small incision from the lumbar L3 to the sacral S1 vertebra was made with a fine scalpel after removing the hair.

  5. Spread the tissue underneath the skin with an agricola retractor, and scratch off the muscle lying on the transverse process of the lumbar L5 vertebra (highlighted in green) with micro spatula to keep it visible (Figure 3, Video 1). Normally the transverse process of the lumbar L5 vertebra is located underneath the muscle within 5 mm from the tip of the iliac crest (Figure 3, left).


    Figure 3. Exposure of the transverse process of the lumbar L5 vertebra. The transverse process of the lumbar L5 vertebra was exposed by scratching off the muscle.

    Video 1. Spinal nerve injury. This video shows how the L4 spinal nerve injury is performed.


  6. Drill the root of the transverse process of the lumbar vertebra at the blue line at a speed of 3,000 rpm (Figure 4) and remove it.


    Figure 4. Removal of the transverse process of the lumbar L5 vertebra. The transverse process of the lumbar L5 vertebra (highlight in green) needs to be removed after drilling the root of it (highlight in blue).

  7. Expose the parallel-lying L3 and L4 spinal nerves by removing the paraspinal muscle and fat from the L5 transverse process with forceps (Figure 5).


    Figure 5. Exposure of L4 spinal nerve. L4 spinal nerve was exposed by removing the paraspinal muscle and fat from the L5 transverse process.

  8. Carefully isolate the L4 spinal nerve with fine forceps and cut it with spring scissors without injuring the L3 nerve (Figure 6).


    Figure 6. Cut of L4 spinal nerve. L4 spinal nerve was cut with spring scissors.

  9. After making sure there is no bleeding from the lesion site (see Notes), suture the wound and the surrounding skin with 5-0 silk sutures (Figure 7). Keep the mouse on a heating pad after the operation until the mouse wakes up (Figure 7).


    Figure 7. Suturing the wound. The wound and the surrounding skin were sutured, and the mouse was placed on a heating pad.

Notes

  1. To avoid causing undesirable tissue inflammation at the operation site, it is important to try to keep the operative time as short as possible; normally this procedure takes approximately 7 min to complete in our laboratory.
  2. If there is accidental bleeding from the operation site, apply proper pressure with a cotton bud. If the bleeding does not stop, the mouse should not be used for further experiments.
  3. Since there are strain-dependent variations in the number of lumbar vertebrae in mice (Rigaud et al., 2008), careful identification and isolation of the L4 spinal nerve is necessary.
  4. As a control, sham surgery could be performed by exposing and drilling the root of the transverse process of the lumbar vertebra without removing the process to keep the L4 spinal nerve intact.

Acknowledgments

This protocol has been developed based on a previously published procedure (Kim and Chung, 1992; Rigaud et al., 2008) with some modifications. T.M. is granted as a JSPS postdoctoral fellowship for research abroad. This work was supported by Grant-in Aid of for Scientific Research (KAKEN 25117013, 15H02522, 16K18885) from JSPS (to K.I., M.T., Y.K.).

References

  1. Burma, N. E., Leduc-Pessah, H., Fan, C. Y. and Trang, T. (2016). Animal models of chronic pain: Advances and challenges for clinical translation. J Neurosci Res 95(6):1242-1256.
  2. Kim, S. H. and Chung, J. M. (1992). An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. Pain 50(3): 355-363.
  3. Masuda, T., Ozono, Y., Mikuriya, S., Kohro, Y., Tozaki-Saitoh, H., Iwatsuki, K., Uneyama, H., Ichikawa, R., Salter, M. W., Tsuda, M. and Inoue, K. (2016). Dorsal horn neurons release extracellular ATP in a VNUT-dependent manner that underlies neuropathic pain. Nat Commun 7: 12529. 
  4. Rigaud, M., Gemes, G., Barabas, M. E., Chernoff, D. I., Abram, S. E., Stucky, C. L. and Hogan, Q. H. (2008). Species and strain differences in rodent sciatic nerve anatomy: implications for studies of neuropathic pain. Pain 136(1-2): 188-201. 

简介

神经性疼痛是高度虚弱的慢性疼痛病症之一,目前尚无治疗。为了揭示神经性疼痛的潜在机制,已经建立了各种动物模型(缅甸等人,2016)。该方案描述了如何准备脊髓神经损伤模型(Kim和Chung,1992; Rigaud等人,2008; Masuda等人,2016),其中最大经常使用和高度可重现的模型,其中在外周和中枢神经系统中发生多次改变。

背景 揭示神经性疼痛的潜在机制是开发有效治疗以达到最佳治疗的必要条件。因此,已经开发了用于神经性疼痛的各种动物模型。特别地,啮齿动物模型经常被使用,因为它们是高度可重复性的,并且在神经性疼痛患者中也观察到疼痛超敏反应。在本协议中,我们描述如何准备小鼠脊髓神经损伤模型。

关键字:神经性疼痛, 神经损伤, 脊髓, 小鼠, 背根神经节(DRG), 触诱发痛

材料和试剂

  1. 5-0丝线缝合(Alfresa Pharma,目录号:GA05SW)
  2. 无菌手术刀(FATHER Safety Razor,目录号:No.10)
  3. 野生型C57BL/6小鼠(6-15周龄)(日本Clea)
  4. 异氟烷(Mylan)
  5. 75%乙醇(Takasugi Pharmaceutical)

设备

  1. 电动剪刀(大东电机行业,目录号:605AD,特价C)
    注意:本产品已停产。
  2. Agricola牵开器(Fine Science Tools,目录号:17005-04)
  3. 镊子(NATSUME SEISAKUSHO,目录号:A-14)
  4. 万纳斯春剪(精细科学工具,目录号:15070-08)
  5. 电钻(URAWA Kogyo,型号:MINITORJET UC210)
  6. 手术刀架(NATSUME SEISAKUSHO,目录号:D-11)
  7. 加热垫(VIVARIA,目录号:MP-916-NV)
  8. 双面微型铲(Fine Science Tools,目录号:10091-12)
  9. 异氟烷分配器(FORWICK,MURACO Medical)

程序

道德要遵循关于动物住房和动物实验的当地动物伦理学。

  1. 为了诱导麻醉,将鼠标放在塑料室中,其中通过管将5%异氟烷提供给分配器(图1)。大约需要30秒


    图1.用异氟烷进行吸入麻醉 使用2-3%异氟烷通过吸入口罩麻醉小鼠。

  2. 通过设置在板上,将头部放入与分配器相连的面罩中,将2-3%吸入异氟烷麻醉(图1)。
  3. 通过用镊子(脚趾捏)施加压力,确保鼠标充分麻醉。
  4. 用电动剪刀将头发从鼠标背部的正确区域取出,并用75%乙醇清洗后,用腰部L3至骶骨S1椎骨的细小手术刀进行小切口(图2)。


    图2.背部皮肤上的小切口从头部L3到骶骨S1椎骨的小切口在脱毛后用精细的手术刀做成。

  5. 用农用牵引器将组织覆盖在皮肤下方,并用微型刮刀刮掉位于腰椎L5椎骨横向过程中的肌肉(以绿色突出显示),以保持其可见(图3,视频1)。通常,腰椎L5椎骨的横向过程位于距离髂嵴顶部5mm以内的肌肉的下方(图3左侧)。


    图3.腰椎L5椎骨横向过程的暴露腰椎L5椎骨的横向过程是通过刮伤肌肉而暴露的。

  6. 以3000rpm的速度在蓝线处钻取腰椎横突的根部(图4),并将其移除。


    图4.腰椎L5椎骨的横向过程的去除腰椎L5椎骨的横向过程(绿色突出显示)需要在钻孔的根部(蓝色突出显示)后被移除。

  7. 通过用镊子从L5横向过程中除去椎旁肌肉和脂肪,从而暴露平行L3和L4脊神经(图5)。


    图5. L4脊髓神经的暴露通过从L5横向过程中去除脊椎旁肌肉和脂肪来暴露L4脊神经。

  8. 用精细镊子仔细分离L4脊神经,用弹簧剪刀切割,不会伤害L3神经(图6)。


    图6. L4脊髓神经切断用弹簧剪刀剪下L4脊髓神经
  9. 确保病变部位没有出血(见注意事项),用5-0丝线缝合伤口和周围皮肤(图7)。操作后将鼠标放在加热垫上,直到鼠标醒来(图7)

    图7.缝合伤口缝合伤口和周围皮肤,将小鼠放在加热垫上。
  10. 笔记

    1. 为了避免在手术部位引起不希望的组织炎症,重要的是尽可能保持手术时间尽可能短。通常这个程序大约需要7分钟才能在我们的实验室完成。
    2. 如果手术部位意外出血,应用棉花芽施加适当的压力。如果出血不止,小鼠不应该用于进一步的实验。
    3. 由于小鼠中腰椎数量存在应变依赖性变化(Rigaud等,2008),需要仔细鉴别和分离L4脊神经。
    4. 作为对照,假手术可以通过暴露和钻孔腰椎横突的根部进行,而不需要去除保持L4脊神经完整的过程。

    致谢

    该协议已经基于以前公布的程序(Kim和Chung,1992; Rigaud等人,2008)而进行了一些修改。 T.M.被授予JSPS博士后研究奖学金。这项工作得到JSPS(至K.I.,M.T.,Y.K。)的科学研究助学金(KAKEN 25117013,15H02522,16K18885)的支持。

    参考

    1. 缅甸,NE,Leduc-Pessah,H.,Fan,CY和Trang,T。(2016)。慢性疼痛的动物模型:临床翻译的进展和挑战 J Neurosci Res 95(6):1242-1256。
    2. Kim,SH和Chung,JM(1992)。  An大鼠节段性脊髓神经连接产生的外周神经病变的实验模型。 50(3):355-363。
    3. Masuda,T.,Ozono,Y.,Mikuriya,S.,Kohro,Y.,Tozaki-Saitoh,H.,Iwatsuki,K.,Uneyama,H.,Ichikawa,R.,Salter,MW,Tsuda,M.和Inoue,K.(2016)。背角神经元以VNUT依赖的方式释放细胞外ATP,作为神经性疼痛的基础。 7:12529. 
    4. Rigaud,M.,Gemes,G.,Barabas,ME,Chernoff,DI,Abram,SE,Stucky,CL和Hogan,QH(2008)。  啮齿动物坐骨神经解剖学中的物种和应变差异:对神经性疼痛研究的影响 疼痛 136(1-2):188-201。 
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用:Masuda, T., Kohro, Y., Inoue, K. and Tsuda, M. (2017). Peripheral Nerve Injury: a Mouse Model of Neuropathic Pain. Bio-protocol 7(9): e2252. DOI: 10.21769/BioProtoc.2252.
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