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Spared Nerve Injury Model of Neuropathic Pain in Mice
小鼠神经病理性疼痛的保留性神经损伤模型   

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本实验方案简略版
Nature Neuroscience
Aug 2017

 

Abstract

Experimental models of peripheral nerve injury have been developed to study mechanisms of neuropathic pain in living animals. The spared nerve injury (SNI) model in rodents is a partial denervation model, in which the common peroneal and tibial nerves are injured, producing consistent and reproducible tactile hypersensitivity in the skin territory of the spared, intact sural nerve. SNI-operated mice require less force applied to the affected limb to elicit a withdrawal behavior as compared to sham mice. This effect is observed as early as 2 days after surgery and lasts for at least 1 month. We describe detailed surgical procedures to establish the SNI mouse model that has been widely used for investigating mechanisms of neuropathic pain.

Keywords: Mouse pain model (小鼠疼痛模型), SNI surgery (SNI手术), Peripheral nerve injury (坐骨神经), Neuropathic pain (神经病理性疼痛)

Background

Partial nerve injury animal models have been developed for the purpose of studying the molecular, cellular, and circuit mechanisms of neuropathic pain (Bennett and Xie, 1988; Seltzer et al., 1990; Kim and Chung, 1992). A partial denervation model enables researchers to investigate structural and functional changes in diverse groups of neuronal and non-neuronal cells. Studies can be performed during the initiation, progression (also known as acute) and maintenance (chronic) phases of neuropathic pain, as well as at different anatomical sites along the pain pathway including distal vs. proximal peripheral nerve fibers, dorsal root ganglion, spinal cord, subcortical and cortical areas. The spared nerve injury (SNI) model involves partial nerve injury where the common peroneal and tibial nerves are injured, producing consistent and reproducible pain hypersensitivity in the territory of the spared sural nerve (Decosterd and Woolf, 2000; Shields et al., 2003). This model has proved to be robust, demonstrating substantial and prolonged changes in behavioral measures of mechanical sensitivity and thermal responsiveness (Bourquin et al., 2006). These features closely mimic the cardinal symptoms of clinically described neuropathic pain disorders.

Materials and Reagents

  1. Cotton-wool applicator
  2. Double edge razor blades (Baili, catalog number: BP005 )
  3. Povidone-Iodine Prep Pad (Dynarex, catalog number: 1108 )
  4. 6-0 nylon suture (Surgical Specialties, Look, catalog number: 916B )
  5. 8-0 nylon suture (Fine Science Tools, catalog number: 12051-08 )
  6. C57BL/6J male mice, 8-12 weeks of age (THE JACKSON LABORATORY, catalog number: 000664 )
  7. Sterile Lubricant Eye Ointment (Stye)
  8. Ketamine hydrochloride (Ketathesia, NDC 11695-0702-1)
  9. Xylazine Sterile Solution (AnaSed, NDC 59399-110-20)
  10. Sterile saline
  11. Ketamine and xylazine (KX) mixture (see Recipes)

Equipment

  1. Stereomicroscope (Olympus, model: SZX10 )
  2. LED surgical light (Schott ACE light source with EKE lamp, Schott, model: A20500 )
  3. Dissecting scissors and forceps (Fine Science Tools, catalog numbers: 14094-11 , 14084-09 , 15000-08 , 11150-10 )
  4. Fine forceps (Fine Science Tools, catalog number: 11253-20 )
  5. Vannas spring scissors (Fine Science Tools, catalog number: 15000-08 )
  6. Electronic von Frey Anesthesiometer (IITC Life Science, catalog number: 2392 )

Procedure

Note: All procedures in this study were approved by the New York University School of Medicine Institutional Animal Care and Use Committee (IACUC) as consistent with the National Institute of Health (NIH) Guide for the Care and Use of Laboratory Animals to ensure minimal animal use and discomfort.

  1. Spared nerve injury surgery
    1. Anesthetize mice with a mixture of KX (0.1 ml/20 g mouse, intraperitoneal injection).
      Note: Assess depth of anesthesia with hindlimb or tail pinch. An animal deeply anesthetized does not react to stimulus. The mouse should be placed on a heating blanket for the maintenance of normothermia while undergoing anesthesia.
    2. Apply ophthalmic ointment to the eyes with a cotton-wool applicator.
    3. Shave the skin on the lateral surface of the left thigh using a razor blade (Figure 1A) followed by topical application of povidone-iodine prep pad.
    4. Make a single, small skin incision at the mid-thigh level with fine scissors (#14094-11) using the femur as a landmark (Figure 1B) and make blunt dissection using the dull portion of the dissection scissors (# 14084-09) through the biceps femoris muscle (BFM) (Figure 1C). Expose the sciatic nerve and its three branches (Figure 1D).
      Note: Perform minimal retraction when exposing the sciatic nerve and its three branches. If there is accidental bleeding from the operation site, apply proper pressure with a cotton bud until coagulation. If bleeding persists, the mouse should not be used for further experiments.
    5. For the SNI operation, distal to the trifurcation of the sciatic nerve, ligate the common peroneal and tibial nerves using 8-0 nylon suture (Figures 1E and 1F) and axotomize with Vannas spring scissors (#15000-08), removing a 2-4 mm piece of each distal nerve stump (Figure 1G). Keep the sural nerve intact (Figures 1E-1G). Avoid any stretching or contact with the spared sural nerve. In the sham operation, the aforementioned manipulations of the sciatic nerve and its branches are not performed.
    6. Close incisions with muscle and skin sutures (Figure 1H).
    Note: The SNI surgery can be performed in both mice and rats for the study of neuropathic pain.


    Figure 1. Spared nerve injury surgical procedure to induce neuropathic pain in mice. A. Mouse was anesthetized with KX and positioned prone. Surgical area was then shaved and disinfected. The paw was abducted and elevated from the table. B. White line indicates the incision site on left hindlimb or thigh. C. Following the incision along the white line, the biceps femoris muscle (BFM) was exposed and a careful blunt dissection was made through to expose the trifurcation of the sciatic nerve. D. Exposure of the sciatic nerve and peripheral branches: common peroneal (CPN), tibial (TN) and sural nerves (SN). E. An 8-0 nylon suture was passed under the common peroneal and tibial nerves. F. Ligation of the common peroneal and tibial nerves was performed with a surgical knot. G. The ligated nerves were transected distally and a 2 mm section was removed to prevent nerve regeneration. The surgical steps in panels E-G were not performed in the sham operation. Care was taken to avoid contact with the sural nerve. Scale bar = 2 mm. H. Muscles were reapproximated, followed by overlying skin. The skin was closed with 6-0 nylon suture with at least 3 individual knots along the incision.

    1. The SNI-operated animals should have normal food intake, growth, display regular movements, and grooming.
    Note: Behavior testing can be performed immediately following recovery from anesthesia.

  2. Behavior testing
    The von Frey test is used to assess the onset and maintenance of mechanical allodynia over time.
    1. Animals were placed in clear plexiglass cages on an elevated mesh floor and tested after 30 min of habituation (Figure 2A).
      Note: During the 30-min habituation before behavior testing, place a small amount of food in testing chambers to help the mice readjust to a new environment, which also lessens their general activity.
    2. In all animal groups, mechanical paw withdrawal threshold was examined using an electronic von Frey anesthesiometer (Figure 2B) with #8 flexible von Frey hair which delivers force up to 11 g (Figure 2C). The anesthesiometer displays the actual force at which paw withdrawal behavior occurs. To perform measurements, first ensure that von Frey hair is securely attached to the anesthesiometer probe. Second, clear the reading on the anesthesiometer before the measurement. Third, direct the von Frey hair through the mesh floor to the lateral plantar aspect (the sural nerve skin territory) of the hind paw (Figure 2D) and record the force displayed.
    3. Three trials of withdrawal per paw were recorded with intervals of 5 min in between measurements. An average was reported for each day tested (as shown in Figure 2E).
      Note: The von Frey test should be performed during the light cycle by the same researcher, who should be blinded to the surgery and treatments.
    4. After SNI, behavioral tests were performed at designated time points (e.g., 2, 7, 14 and 28 days) after surgery (Figure 2E).
      Note: The course of SNI-induced neuropathic pain in mice is usually divided into development (1-7 days) and maintenance (8-14 days) phases. Depending on the purpose of each study, behavior tests should be planned accordingly.


      Figure 2. Measuring hindlimb paw withdrawal threshold before and after SNI. A. SNI and sham-operated mice were placed in plexiglass cages on an elevated mesh platform for paw access. B and C. Electronic von Frey anesthesiometer with #8 von Frey hair. D. Plantar view of the left hindlimb paw from a mouse after SNI operation. The red area on the photograph corresponds to the sural nerve skin territory that was tested with the von Frey hair, while the blue area corresponds to the tibial nerve skin territory, which was denervated from SNI surgery and should not be tested during the test. E. Paw withdrawal threshold measured in grams from ipsilateral and contralateral hindlimbs in both SNI and sham-operated mice over 1 month (Two-way ANOVA followed by Tukey’s test; 2 day: P = 0.025; 7 day: P < 0.001; 14 day: P < 0.001; 31 day: P < 0.001. n = 17 in SNI group, and n = 12 in sham group) (Cichon et al., 2017).

Data analysis

A complete description of statistics used for analyzing von Frey behavioral experiments is presented in Cichon et al. (2017).

Notes

  1. Positive aspects: SNI surgery is a simple procedure to carry out and can be performed by researchers with some surgical experience. Also, following SNI surgery, mice reliably display mechanical hypersensitivity as early as 2 days after injury, and develop long-term hypersensitivity for at least 30 days. Sham-operated mice initially show increased mechanical sensitivity (e.g., 2 days after surgery), which could be related to the surgical inflammation, but should return to baseline levels within days (Figure 2E). Cortical neurons in the awake behaving SNI/sham mice could be imaged with two-photon microscopy (Yang et al., 2013; Cichon et al., 2017). Thus, experiments can be performed to study mechanisms for the initiation, progression and maintenance of neuropathic pain.
  2. Negative aspects: SNI model induces lesions in the peroneal and tibial nerves, leaving the sural nerve intact. Because the sural nerve innervates the skin on the lateral aspect of the hind paw (Figure 2D), experience and repetitive measurements are required to improve the accuracy and precision of paw withdrawal testing.

Recipes

  1. Ketamine and xylazine mixture
    To make 50 ml of KX:
    10 ml ketamine (100 mg/ml)
    7.5 ml xylazine (20 mg/ml)
    32.5 ml of sterile saline (0.9% NaCl), mix well
    Store it away from light exposure and at room temperature

Acknowledgments

This protocol is adapted from the previously published paper (Cichon et al., 2017). This work was supported by National Institutes of Health grants R01GM107469 and R21NS106469 to G.Y. The authors have nothing to disclose.

References

  1. Bennett, G. J. and Xie, Y. K. (1988). A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33(1): 87-107.
  2. Bourquin, A. F., Suveges, M., Pertin, M., Gilliard, N., Sardy, S., Davison, A. C., Spahn, D. R. and Decosterd, I. (2006). Assessment and analysis of mechanical allodynia-like behavior induced by spared nerve injury (SNI) in the mouse. Pain 122(1-2): 14 e11-14.
  3. Cichon, J., Blanck, T. J. J., Gan, W. B. and Yang, G. (2017). Activation of cortical somatostatin interneurons prevents the development of neuropathic pain. Nat Neurosci 20(8): 1122-1132.
  4. Decosterd, I. and Woolf, C. J. (2000). Spared nerve injury: an animal model of persistent peripheral neuropathic pain. Pain 87(2): 149-158.
  5. 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.
  6. Seltzer, Z., Dubner, R. and Shir, Y. (1990). A novel behavioral model of neuropathic pain disorders produced in rats by partial sciatic nerve injury. Pain 43(2): 205-218.
  7. Shields, S. D., Eckert, W. A., 3rd and Basbaum, A. I. (2003). Spared nerve injury model of neuropathic pain in the mouse: a behavioral and anatomic analysis. J Pain 4: 465-470.
  8. Yang, G., Pan, F., Chang, P. C., Gooden, F. and Gan, W. B. (2013). Transcranial two-photon imaging of synaptic structures in the cortex of awake head-restrained mice. Methods Mol Biol 1010: 35-43.

简介

等人,1990; Kim和Chung,1992 )。部分去神经支配模型使研究人员能够研究不同组的神经元细胞和非神经元细胞的结构和功能变化。研究可以在神经性疼痛的起始,进展(也称为急性)和维持(慢性)阶段以及在沿着疼痛途径的不同解剖部位期间进行,包括远侧与近侧周围神经纤维,背根神经节,脊髓绳索,皮质下和皮层区域。备用神经损伤(SNI)模型包括部分神经损伤,其中腓总神经和胫神经受损,在腓肠神经部位产生一致且可再现的疼痛超敏反应(Decosterd和Woolf,2000; Shields等人, ,2003)。该模型已被证明是有力的,证明了机械敏感性和热响应性的行为测量值的实质和长期变化(Bourquin等人,2006)。这些特征与临床描述的神经性疼痛疾病的主要症状密切相似。

关键字:小鼠疼痛模型, SNI手术, 坐骨神经, 神经病理性疼痛

材料和试剂

  1. 棉花涂抹器
  2. 双刃剃须刀(百利,目录号:BP005)
  3. 聚维酮碘预浸垫(Dynarex,目录号:1108)
  4. 6-0尼龙缝线(Surgical Specialties,Look,目录号:916B)
  5. 8-0尼龙缝线(Fine Science Tools,目录号:12051-08)
  6. C-8BL / 6J雄性小鼠,8-12周龄(THE JACKSON LABORATORY,目录号:000664)
  7. 无菌润滑剂眼膏(Stye)
  8. 盐酸氯胺酮(Ketathesia,NDC 11695-0702-1)
  9. 赛拉嗪不育溶液(AnaSed,NDC 59399-110-20)
  10. 无菌生理盐水
  11. 氯胺酮和甲苯噻嗪(KX)混合物(见食谱)

设备

  1. 立体显微镜(奥林巴斯,型号:SZX10)
  2. LED手术灯(带EKE灯的Schott ACE光源,Schott,型号:A20500)
  3. 解剖剪刀和镊子(Fine Science Tools,产品目录号:14094-11,14084-09,15000-08,11150-10)
  4. 细镊子(Fine Science Tools,目录号:11253-20)
  5. Vannas弹簧剪刀(Fine Science Tools,目录号:15000-08)
  6. 电子冯弗雷麻醉计(IITC生命科学,目录号:2392)

程序

注意:本研究中的所有程序均经纽约大学医学院动物管理和使用委员会(IACUC)批准,与国家卫生研究院(NIH)实验动物护理和使用指南一致确保最小的动物使用和不适。

  1. 备用神经损伤手术
    1. 用KX(0.1ml / 20g小鼠,腹膜内注射)混合麻醉小鼠。
      注:麻醉深度用后肢或尾巴捏。深度麻醉的动物不会对刺激产生反应。
      小鼠应置于加热毯上,以保持正常的麻醉状态。

    2. 用棉签涂抹眼药膏
    3. 使用剃刀刀片(图1A)剃去左大腿外侧表面的皮肤,然后局部应用聚维酮碘制备垫。
    4. 使用股骨作为地标(图1B),用精细剪刀(#14094-11)在大腿中部水平切开一个单一的小型皮肤切口,并使用剥离剪刀的无光泽部分(#14084-09)进行钝性解剖,通过股二头肌(BFM)(图1C)。暴露坐骨神经及其三个分支(图1D)。
      注意:暴露坐骨神经及其三个分支时应尽可能缩回。如果手术部位出现意外出血,请用棉签保持适当压力,直至凝固。如果出血仍然存在,小鼠不应该用于进一步的实验。
    5. 对于坐骨神经三叉分支远端的SNI手术,使用8-0尼龙缝线(图1E和1F)结扎腓总神经和胫神经,并用Vannas弹簧剪刀(#15000-08)切断,每个远端神经残端4 mm(图1G)。保持腓肠神经完整(图1E-1G)。避免任何伸展或与免疫腓肠神经接触。在假手术中,不执行上述的坐骨神经及其分支的操作。
    6. 用肌肉和皮肤缝线关闭切口(图1H)。
    注意:可以在小鼠和大鼠中进行SNI手术以研究神经性疼痛。


    图1.用于诱导小鼠神经性疼痛的备用神经损伤外科手术A.用KX麻醉小鼠并定位于俯卧位。然后剃毛并消毒手术区域。爪子从桌子上被绑架并抬高。 B.白线表示左后肢或大腿的切口部位。 C.沿着白线切开后,暴露股二头肌(BFM),并通过小心的钝器解剖以暴露坐骨神经的三叉神经。 D.坐骨神经和周围分支的暴露:腓总神经(CPN),胫骨(TN)和腓肠神经(SN)。 E.将8-0尼龙缝合线穿过腓总神经和胫神经。 F.腓总神经和胫神经的结扎用手术结进行。 G.将结扎的神经向远端横切并移除2mm切片以防止神经再生。 E-G图中的手术步骤未在假手术中进行。小心避免与腓肠神经接触。比例尺= 2毫米。 H.肌肉重新接近,然后覆盖皮肤。
    用6-0尼龙缝线将皮肤封闭,沿切口至少有3个单独的结
    1. SNI手术的动物应该有正常的食物摄入,生长,显示有规律的动作和梳理。
    注:行为测试可在麻醉恢复后立即执行。

  2. 行为测试
    冯弗雷测试用于评估机械异常性疼痛的发作和维持随着时间的推移。
    1. 将动物置于高架网眼底上的透明有机玻璃笼中并在习惯化30分钟后测试(图2A)。
      注意:在行为测试前的30分钟习惯化期间,将少量食物放入测试室以帮助老鼠重新适应新的环境,这也降低了它们的一般活动。
    2. 在所有动物组中,使用具有#8挠性von Frey头发的电子von Frey麻醉仪(图2B)检查机械缩爪阈值,所述vy Frey头发提供高达11g的力(图2C)。麻醉计显示出现爪缩回行为的实际力量。要进行测量,首先确保von Frey头发牢固地连接到麻醉计探头上。其次,在测量前清除麻醉仪上的读数。第三,将von Frey头发穿过网底到达后爪的外侧足底面(腓肠神经区)(图2D)并记录显示的力。
    3. 在两次测量之间记录三次每只脚的退缩试验间隔为5分钟。报告每天测试的平均值(如图2E所示)。
      注意:von Frey测试应该在光周期期间由同一位研究员进行,他们应该对手术和治疗不知情。
    4. 在SNI之后,在手术后的指定时间点(例如,2,7,14和28天)进行行为测试(图2E)。
      注意:SNI诱导的小鼠神经性疼痛过程通常分为发育(1-7天)和维持(8-14天)阶段。根据每项研究的目的,应该相应地规划行为测试。


      图2.在SNI之前和之后测量后肢缩爪阈值A.将SNI和假手术的小鼠放置在高架网状平台上的有机玻璃笼中以便进入爪子。 B和C.电子冯弗雷麻醉与#8冯弗雷头发。 D. SNI手术后小鼠的左后肢足底的视图。照片上的红色区域对应于用von Frey头发测试的腓肠神经皮肤区域,而蓝色区域对应于SNI手术失神经并且在测试期间不应该测试的胫骨神经皮肤区域。 E.在1个月内SNI和假手术小鼠的同侧和对侧后肢以克为单位测量的爪缩回阈值(双向ANOVA,随后Tukey's测试; 2天:P <= em = 0.025; 7天: P <0.001; 14天: <0.001; 31天: <0.001。在SNI组中≥17,在假组中 n = 12)(Cichon等人,2017)。

数据分析

Cichon等人提出了用于分析von Frey行为实验的统计学的完整描述。 (2017)。

笔记

  1. 积极的方面:SNI手术是一个简单的程序,可以由具有一定手术经验的研究人员执行。此外,在SNI手术之后,小鼠早在损伤后2天就可靠地显示机械超敏反应,并且长期超敏反应至少持续30天。假手术小鼠最初表现出机械敏感性增加(例如术后2天,例如术后2天),这可能与手术炎症有关,但应在几天内回复至基线水平(图2E)。清醒行为的SNI / sham小鼠中的皮层神经元可以用双光子显微镜成像(Yang等人,2013; Cichon等人,2017)。因此,可以进行实验来研究神经性疼痛的发生,发展和维持机制。
  2. 消极方面:SNI模型诱导腓骨神经和胫神经损伤,使腓肠神经完好无损。由于腓肠神经支配后爪外侧皮肤(图2D),需要经验和重复测量来提高爪缩回测试的准确性和精确性。

食谱

  1. 氯胺酮和甲苯噻嗪混合物
    要制造50毫升的KX:
    10毫升氯胺酮(100毫克/毫升)
    7.5毫升赛拉嗪(20毫克/毫升)
    32.5毫升无菌生理盐水(0.9%NaCl),充分混合
    将其远离光线照射并在室温下
    存放

致谢

该协议改编自以前发表的论文(Cichon et al。,2017)。这项工作得到了美国国立卫生研究院拨款R01GM107469和R21NS106469对G.Y的支持。作者没有什么可披露的。

参考

  1. Bennett,G.J。和Xie,Y.K。(1988)。 大鼠出现外周性单神经病,引起类似于男性的疼痛感。 疼痛 33(1):87-107。
  2. Bourquin,A.F。,Suveges,M.,Pertin,M.,Gilliard,N.,Sardy,S.,Davison,A.C。,Spahn,D.R。和Decosterd,I.(2006)。 评估和分析小鼠中备用神经损伤(SNI)引起的机械异常性疼痛样行为。 疼痛 122(1-2):14 e11-14。
  3. Cichon,J.,Blanck,T.J.J。,Gan,W.B。和Yang,G。(2017)。 皮质生长抑素中间神经元的激活可防止神经性疼痛的发展。 Nat Neurosci 20(8):1122-1132。
  4. Decosterd,I.和Woolf,C.J。(2000)。 备用神经损伤:持续性周围神经性疼痛的动物模型 疼痛 87(2):149-158。
  5. Kim,S.H。和Chung,J.M。(1992)。 一种由大鼠节段性脊神经结扎产生的周围神经病的实验模型 < 疼痛 50(3):355-363。
  6. Seltzer,Z.,Dubner,R.和Shir,Y。(1990)。 通过部分坐骨神经损伤在大鼠中产生的神经性疼痛障碍的新型行为模型。 疼痛 43(2):205-218。
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Copyright: © 2018 The Authors; exclusive licensee Bio-protocol LLC.
引用:Cichon, J., Sun, L. and Yang, G. (2018). Spared Nerve Injury Model of Neuropathic Pain in Mice. Bio-protocol 8(6): e2777. DOI: 10.21769/BioProtoc.2777.
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Shizhen Luo
University of arizona
Haven't started my experiment.
10/1/2018 9:46:19 AM Reply