Mechanical Allodynia Assessment in a Murine Neuropathic Pain Model

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Apr 2017



Experimental animal models are unique tools (i) to study pain transmission and pathophysiology of neuropathic pain, (ii) to identify novel molecular targets and (iii) to test the potential analgesic effect of specific molecules. The chronic constriction injury (CCI) model of neuropathic pain is the first model of post-traumatic painful peripheral neuropathy, originally developed by Bennett and Xie in the late 1980s. The chronic constriction is performed in the sciatic nerve and induces a partial denervation involving myelinated afferent axons and unmyelinated axons. Damage to unmyelinated axons is much more severe than myelinated afferents. As the model induces a partial denervation, it is very useful for the analysis of pain behaviours. Stimulation of the hind paw, a target of the sciatic nerve, induces pain which can be quantitated. Thus, mechanical allodynia is usually assessed 7, 14 and 21 days after CCI of the sciatic nerve by measuring the hind paw withdrawal response to von Frey filament stimulation. Here, we describe in detail the protocol allowing a reliable and reproducible CCI model in mice. Overall, researchers most commonly use this surgical model to discover more efficacious drugs for the pharmacological control of chronic pain states.

Keywords: Pain (疼痛), Nociception (伤害感受), Neuropathic pain (神经性疼痛), Analgesia (镇痛), Mice (小鼠)


The chronic constriction injury (CCI) model of neuropathic pain was first developed by Bennett and Xie (1988). The chronic constriction is applied to the sciatic nerve mimicking a post-traumatic painful peripheral neuropathy. This model induces a partial denervation and, therefore, is very useful for a quantitative analysis of pain behaviours and for the evaluation of analgesic effect of novel drugs. The CCI of the sciatic nerve is carried out under isoflurane anesthesia (5% for induction and 2% for maintenance). The biceps femoris and the gluteus superficialis are separated by dissection to expose the sciatic nerve. The CCI is induced by loosely tying one ligature around the sciatic nerve, to preserve epineural circulation.

Analysis of pain behaviour is assessed by measuring mechanical allodynia 7, 14 and 21 days after surgery. Interestingly, one of the advantages of the CCI model is the objective score of pain behaviour in response to von Frey stimulation. Mechanical allodynia is quantitated by measuring the hind paw withdrawal response to von Frey filament stimulation. Mice are placed in a dark box with a wire grid bottom through which the von Frey filaments are applied by using the up-down paradigm previously described (Chaplan et al., 1994). Lack of response to a filament indicates the next filament with a higher bending force in the following stimulation, whereas a positive response indicated the next filament with a lower bending force. Each filament is applied and pressed perpendicularly to the plantar surface of the hind paw until it bends. The filament that evokes 3 paw withdrawals is assigned as the pain threshold in grams. Mice are treated with vehicle, novel potential analgesic drugs, or a classical analgesic drug, as positive analgesic control, and the mechanical thresholds are quantified. This protocol has been recently implemented by our research team (Font et al., 2017).

Overall, CCI model and measurements of mechanical allodynia could be considered as an experimental approach to assess the analgesic activity of any potential drug. Thus, we provide here a complete description of the CCI models and assessment of mechanical allodynia aiming at facilitating its implementation by other scientists.

Materials and Reagents

  1. Sterile gauze
  2. Syringes. BD Micro-FineTM Demi, U-100 Insulin, 30 G x ½’’–0.33 x 8 mm (BD, catalog number: 324826 )
  3. Eppendorf tubes (Eppendorf, catalog number: 0030120086 )
  4. Scalpel blades (Sigma-Aldrich, catalog number: S2646 )
  5. 6-0 silk suture (Ethicon, catalog number: MCP492G )
  6. 5-0 Dermalon suture (Covidien, catalog number: 1756-21 )
  7. Animals
    Adult C57BL/6J male mice (Charles River, Calco, Italy) weighing 20-25 g are used. Six-week-old male mice are allowed to habituate in the animal room for 2 weeks
    Note: All animals are housed in groups of five in standard cages with access to food and water ad libitum, and maintained under 12 h dark/light cycle (starting at 7:30 AM), 22 °C temperature, and 66% humidity. All manipulations are carried out between 9:00 and 16:00 h. Procedures were performed in accordance with relevant guidance from the National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23), the Guide for the Care and Use of Laboratory Animals (Clark et al., 1997) and European Union directives (2010/63/EU). The ethics committees of the IRCCS Neuromed Institute and the Italian Ministry of Health approved the protocol. All efforts were made to minimize suffering and reduce the number of animals used in the experiments. To reach statistical significance, at least 10 animals per group are highly recommended.
  8. Isoflurane (Sigma-Aldrich, catalog number: Y0000858 )
  9. Potential analgesic drug to be tested
    Note: In the example provided here we used raseglurant, a negative allosteric modulator of the metabotropic glutamate type 5 (mGlu5) receptor (Font et al., 2017).
  10. Vehicle
    Sterile physiological saline (0.9% NaCl) solution (B. Braun Melsungen, catalog number: 3570380 )
  11. Classical analgesic drugs for comparative purposes
  12. Sterile cleaning solution
    Note: A 70% ethanol in H2O was used.


  1. Surgical instruments, surgery bench, inhalation anaesthesia apparatus
  2. Ugo Basile Gas Anaesthesia (Ugo Basile, catalog number: 22100 ), Ugo Basile Induction Box 7900 (Ugo Basile, catalog number: 7900/10 ) (Figure 1)

    Figure 1. Apparatus for inhalation anaesthesia in mice and rats with an induction chamber (25 x 13 x 13 cm)

  3. Surgical Microscope Leica Wild M650 (Leica, model: Leica Wild M650) (Figure 2)

    Figure 2. Stereomicroscope with an anaesthetized mouse ready to undergo CCI

  4. Fine watchmaker forceps (Sigma-Aldrich, catalog number: T4537-1EA )
  5. Curved blunt-tipped forceps (Sigma-Aldrich, catalog number: T4787-1EA )
  6. Micro-dissecting scissor (Sigma-Aldrich, catalog number: S3146-1EA )
  7. Mathieu needle holder (Fine Science Tools, catalog number: 12510-14 )
  8. Balances (Sartorius, models: CP124S and BL1500S )
  9. von Frey filaments (North Coast Medical, Inc., San Jose, CA, USA) (Figure 3)

    Figure 3. von Frey filaments used to assess mechanical allodynia


  1. Surgical procedure
    1. Anaesthetize mice in an induction chamber by inhalation of 2.5% isoflurane in N2O/O2 (70:30), maintained at 2% by a facemask throughout surgery.
    2. Place mouse under a stereomicroscope, remove hair in the right or left hind leg and sterilize the area by the use of a sterile gauze with 70% alcohol.
    3. Place the mouse in horizontal position with the femur rotated by 90° with the aid of masking tape placed on the foot (Figure 4).

      Figure 4. Mouse placed in horizontal position with the right leg rotated at 90° with the aid of masking tape placed on the foot

    4. Make an incision in the skin and separate the muscles between the gluteus superficialis and the biceps femoris by cutting the connective tissue (Figure 5).

      Figure 5. Separation of muscles gluteus superficialis and biceps femoris by cutting the connective tissue

    5. Move the two muscles to uncover the sciatic nerve.
    6. Using curved blunt-tipped forceps and micro-scissors, softly free the sciatic nerve (proximal to the sciatic trifurcation) from the surrounding connective tissue.
    7. Under a dissecting microscope, make one ligature, proximal to the nerve trifurcation, while taking care to preserve epineural circulation, around the sciatic nerve with 6-0 silk (Figure 6). The ligature has to be tied loosely around the nerve, until it elicits a brief twitch in the respective hind limb, which prevents over-tightening of the ligation.

      Figure 6. Under the dissecting microscope, free the sciatic nerve by curved blunt-tipped forceps and make one ligature with 6-0 silk

    8. Stitch the muscle layer.
    9. Clean the incision and close the skin with 2-3 ligatures of 5-0 dermalon. Apply an iodine solution (Rodine, 10% solution) to disinfect the wound.
    10. Return the mouse to its home cage and check routinely for 72 h.
    11. Clean all the material used.

  2. Mechanical allodynia measurement
    1. Handle mice daily in order to reduce stress.
    2. Prior to behavioural testing, mice need to be habituated to the testing procedure and the restricted area. The testing environment should be kept quiet and well controlled, with constant temperature and humidity levels. Testing sessions should be carried out at the same time (9:00-14:00).
    3. Reagents preparation (weight the analgesic drug and dissolve in vehicle).
    4. Remove the mouse from the house cage and measure its body weight using a balance.
    5. Administration of the vehicle or analgesic drug.
      Administer (i.e., intraperitoneally, i.p.) the analgesic drug to be tested (i.e., raseglurant) or the same volume of vehicle (physiological saline solution) using an insulin syringe.
      Note: The amount (400-500 μl) of drug to be administered should be adjusted according to the dose to be tested (i.e., 10 mg/kg of raseglurant) and the mouse body weight (20-25 g). For example, for a mouse of 20 g of weight we will administer (i.p.) 400 μl of a 500 mg/L raseglurant solution in saline. In addition, all animal experimentation should be carried out by a researcher blind to drug treatments.
    6. Place the mouse in a plastic cylinder placed on a wire mesh table. Habituate for 15 min in cylinders prior to testing to allowing mouse to stay calm and still.
    7. After 20 min, assess mechanical pain thresholds by means of von Frey filaments (Figure 3). Thus, apply filament, bending force range from 0.008 to 3.5 g, to the mid-plantar surface of the hind paw (Figure 7).

      Figure 7. Quantification of mechanical thresholds by the von Frey filaments

    8. Apply each filament, starting from the one with a bending force of 0.008 g, and press it perpendicularly to the plantar surface of the hind paw until it bent for five times over a total period of 30 sec (approximately 2 sec per stimulus) and measure the mouse leg withdrawal after each application (Chaplan et al., 1994). Repeat this procedure 5 times with a 3-min interval. Response in 3 out of 5 stimuli is considered as a positive reaction and the filament is assigned as the pain threshold in grams. Lack of response to a filament indicated the next filament with a higher bending force in the following stimulation, whereas a positive response indicated the next filament with a lower bending force.
    9. Return the mouse to its home cage.
    10. Clean all the material used.

Data analysis

Representative example of data illustrating the type of results obtained is provided below.

  1. Administration of the analgesic drug (i.e., raseglurant) significantly increased pain thresholds in CCI mice (Figure 8).
  2. The results are analyzed either by Student’s t-test or by one-way analysis of variance (ANOVA) followed by a post hoc test, according to the experimental paradigm. P values < 0.05 are considered significant.

    Figure 8. Representative results. Antinociceptive effect of raseglurant (10 mg/kg) as analgesic drug using the CCI of the sciatic nerve animal model. Mechanical allodynia was measured in 21 days post-surgery CCI mice. Thus, animals were intraperitoneally injected with vehicle (Veh, saline) or raseglurant (Ras, 10 mg/kg) and 20 min later the mechanical thresholds were assessed using the von Frey filaments. Values are means ± SEM of 7-9 mice per group. **P < 0.001 Student’s t-test when compared to the vehicle (physiological saline solution) treated animals. Extracted from Font et al., 2017.


This work was supported by ERANET Neuron project ‘LIGHTPAIN’. This protocol was adapted from previous work: Font et al., 2017. The authors declare not conflict of interest.


  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. Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M. and Yaksh, T. L. (1994). Quantitative assessment of tactile allodynia in the rat paw. J Neurosci Methods 53(1): 55-63.
  3. Clark, J. D., Gebhart, G. F., Gonder, J. C., Keeling, M. E. and Kohn, D. F. (1997). Special report: The 1996 guide for the care and use of laboratory animals. ILAR J 38(1): 41-48.
  4. Font, J., Lopez-Cano, M., Notartomaso, S., Scarselli, P., Di Pietro, P., Bresoli-Obach, R., Battaglia, G., Malhaire, F., Rovira, X., Catena, J., Giraldo, J., Pin, J. P., Fernandez-Duenas, V., Goudet, C., Nonell, S., Nicoletti, F., Llebaria, A. and Ciruela, F. (2017). Optical control of pain in vivo with a photoactive mGlu5 receptor negative allosteric modulator. Elife 6.


实验动物模型是独特的工具:(i)研究神经性疼痛的疼痛传递和病理生理学,(ii)鉴定新的分子靶标和(iii)测试特定分子的潜在镇痛作用。神经性疼痛的慢性缩窄性损伤(CCI)模型是最初由Bennett和Xie在二十世纪八十年代后期开发的创伤后疼痛性周围神经病的第一个模型。慢性收缩是在坐骨神经中进行的,并诱导部分去神经支配,包括有髓鞘的传入轴突和无髓鞘轴突。对无髓鞘轴突的损伤比有髓神经传入者严重得多。由于该模型导致部分去神经支配,对疼痛行为的分析非常有用。刺激后爪(坐骨神经的靶标)引起可被定量的疼痛。因此,通常通过测量对von Frey丝刺激的后爪缩回反应,在坐骨神经的CCI后7,14和21天评估机械异常性疼痛。在这里,我们详细描述协议允许在小鼠中可靠和可重复的CCI模型。总的来说,研究人员最常使用这种手术模式来发现更有效的慢性疼痛状态的药物控制药物。


通过在手术后7,14和21天测量机械性异常疼痛来评估疼痛行为的分析。有趣的是,CCI模型的优点之一就是对von Frey刺激的疼痛行为的客观评分。机械性异常性疼痛通过测量对von Frey丝刺激的后爪缩回反应来定量。将小鼠放置在具有线栅底部的黑盒中,通过使用前面描述的上 - 下范例(Chaplan等人,1994),将von Frey细丝施加到线盒底部。缺乏对灯丝的响应表明在接下来的刺激中具有较高的弯曲力的下一根灯丝,而正响应表示具有较低的弯曲力的下一根灯丝。将每根细丝施加并垂直压在后爪的足底表面,直到其弯曲。引起3爪拔出的细丝被指定为以克为单位的疼痛阈值。将小鼠用载体,新型潜在镇痛药或经典镇痛药作为阳性镇痛控制剂,并对机械阈值进行量化。该协议最近已经由我们的研究团队实施(Font等人,2017年)。


关键字:疼痛, 伤害感受, 神经性疼痛, 镇痛, 小鼠


  1. 无菌纱布
  2. 注射器。 BD Micro-FineTM Demi,U-100胰岛素,30 G x½“' - 0.33 x 8 mm(BD,目录号:324826)
  3. Eppendorf管(Eppendorf,目录号:0030120086)
  4. 手术刀片(西格玛奥德里奇,目录号:S2646)
  5. 6-0丝线(Ethicon,目录号:MCP492G)
  6. 5-0 Dermalon缝合(Covidien,目录号:1756-21)
  7. 动物
    使用体重为20-25g的成年C57BL / 6J雄性小鼠(Charles River,Calco,Italy)。
    六周大的雄性小鼠可以在动物房里习惯2周 注意:所有的动物在标准笼内饲养5只,可自由获取食物和水,并在12小时黑暗/光照周期(从上午7:30开始),22℃的温度和66 %湿度。所有的操作都在9:00到16:00之间进行。程序按照国家卫生研究院关于实验动物护理和使用指南(NIH出版物第80-23号),实验动物护理和使用指南(Clark等,1997)的相关指导进行)和欧盟指令(2010/63 / EU)。 IRCCS Neuromed研究所和意大利卫生部伦理委员会批准了该协议。所有的努力都是为了尽量减少痛苦,减少实验中使用的动物的数量。为了达到统计学显着性,强烈推荐每组至少10只动物。
  8. 异氟醚(Sigma-Aldrich,目录号:Y0000858)
  9. 潜在的镇痛药物被测试
    注意:在这里提供的例子中,我们使用了代谢型谷氨酸5型(mGlu5)受体的负变构调节剂raseglurant(Font et al。,2017)。
  10. 车辆
  11. 用于比较目的的古典镇痛药物
  12. 无菌清洁解决方案
    注意:使用H 2 O中的70%乙醇。


  1. 手术器械,手术台,吸入麻醉设备
  2. Ugo Basile Gas Anesthesia(Ugo Basile,产品目录号:22100),Ugo Basile Induction Box 7900(Ugo Basile,产品目录号:7900/10)(图1)

    图1.小鼠和大鼠吸入麻醉的设备(25 x 13 x 13厘米)
  3. Leica Wild M650手术显微镜(Leica,型号:Leica Wild M650)(图2)


  4. 精密制表钳(西格玛奥德里奇,目录号:T4537-1EA)
  5. 弧形钝头钳(西格玛奥德里奇,目录号:T4787-1EA)
  6. 微型解剖剪(西格玛奥德里奇,目录号:S3146-1EA)
  7. Mathieu持针器(精细科学工具,目录号:12510-14)
  8. 天平(赛多利斯,型号:CP124S和BL1500S)
  9. 冯弗雷丝(北海岸医疗公司,圣何塞,加利福尼亚州,美国)(图3)



  1. 手术过程
    1. 在N 2 O / O 2(70:30)中通过吸入2.5%异氟醚麻醉诱导室中的小鼠,在整个手术中通过面罩保持2%。
    2. 将鼠标置于立体显微镜下,在右后腿或左后腿取出头发,并使用含70%酒精的无菌纱布消毒该区域。
    3. 将鼠标置于水平位置,借助放置在脚上的遮蔽胶带将股骨旋转90°(图4)。


    4. 通过切割结缔组织(图5),在皮肤上切开皮肤,并分离臀部表浅肌和股二头肌之间的肌肉。

      图5.通过切割结缔组织 分离肌肉表浅肌肉和肱二头肌

    5. 移动两块肌肉,揭开坐骨神经。
    6. 使用弯曲的钝头钳和微型剪刀,从周围的结缔组织中轻柔地释放坐骨神经(靠近坐骨三叉)。
    7. 在解剖显微镜下,用6-0丝线在坐骨神经周围注意保持神经周细胞循环,在靠近神经分叉处做一个结扎(图6)。必须将绑扎松散地缠绕在神经周围,直到它在相应的后肢中引起短暂的抽搐,这可以防止结扎过度紧缩。


    8. 缝合肌肉层。
    9. 清洁切口,并用2-3个5-0的皮肤纹线结束皮肤。
    10. 将鼠标放回家中,常规检查72小时。
    11. 清洁所有使用的材料。

  2. 机械性异常性疼痛测量
    1. 每天处理小鼠以减轻压力。
    2. 行为测试之前,小鼠需要习惯于测试程序和限制区域。测试环境应保持安静,控制良好,温度和湿度不变。测试会议应同时进行(9:00-14:00)。
    3. 试剂准备(称重止痛药并溶解在载体中)。
    4. 从家里的笼子里取出鼠标,用天平测量它的体重。
    5. 施用载体或镇痛药物。
      注意:给药量(400-500μl)应根据待测试的剂量(即10mg / kg的抗氧化剂)和小鼠的体重(20-25g)进行调整。例如,对于体重为20克的小鼠,我们将给予(i.p.)400微升500毫克/升的生理食盐水溶液。另外,所有的动物实验都应该由盲人进行药物治疗的研究人员进行。
    6. 将鼠标放在放置在金属丝网桌上的塑料圆筒中。
    7. 20分钟后,通过冯弗雷丝评估机械痛阈(图3)。因此,将长丝的弯曲力范围从0.008到3.5克施加到后爪的跖面(图7)。

      图7. von Frey细丝的机械阈值定量

    8. 从0.008克的弯曲力开始,将每根细丝涂上,并垂直按压到后爪的足底表面,直到在30秒的总时间内(每次刺激大约2秒)弯曲五次,然后测量在每次施用后小鼠腿撤回(Chaplan等人,1994)。重复此过程5次,间隔3分钟。响应在5个刺激中的3个被认为是阳性反应,并且灯丝被指定为以克为单位的疼痛阈值。缺乏对灯丝的响应表明在接下来的刺激中具有较高弯曲力的下一根灯丝,而正响应则表明下一根灯丝具有较低的弯曲力。
    9. 将鼠标返回到它的家笼子。
    10. 清洁所有使用的材料。



  1. 给予止痛药(即,, ),显着增加CCI小鼠的疼痛阈值(图8)。
  2. 根据实验范例,通过Student's t检验或通过单向方差分析(ANOVA),然后进行事后检验来分析结果。 P 值&lt; 0.05被认为是重要的。

    图8.代表性的结果使用坐骨神经动物模型的CCI作为镇痛药物的抗阴离子效应(10mg / kg)。在术后21天的CCI小鼠中测量机械异常性疼痛。因此,给动物腹膜内注射载体(Veh,盐水)或者抗氧化剂(Ras,10mg / kg),20分钟后,使用von Frey细丝评估机械阈值。数值是每组7-9只小鼠的平均值±SEM。 ** P 0.001与运载体(生理盐水溶液)处理的动物相比较的学生的测试。
    摘自Font et。,2017。


这项工作得到了ERANET Neuron项目“LIGHTPAIN”的支持。该协议是从以前的工作改编的:Font et al。,2017。作者声明没有利益冲突。


  1. Bennett,G.J。和Xie,Y.K。(1988)。 大鼠的外周性单神经病,会产生类似于人类所见的疼痛感觉障碍。 疼痛 33(1):87-107。
  2. Chaplan,S.R.,Bach,F.W.,Pogrel,J.W.,Chung,J.M。和Yaksh,T.L。(1994)。 大鼠爪中触觉异常性疼痛的定量评估 Neurosci Methods 53(1):55-63。
  3. Clark,J.D。,Gebhart,G.F.,Gonder,J.C。,Keeling,M.E。和Kohn,D.F。(1997)。 特别报告:1996年实验动物护理和使用指南 ILAR J 38(1):41-48。
  4. 字体,J.,Lopez-Cano,M.,Notartomaso,S.,Scarselli,P.,Di Pietro,P.,Bresoli-Obach,R.,Battaglia,G.,Malhaire,F.,Rovira,X。 Catena,J.,Giraldo,J.,Pin,JP,Fernandez-Duenas,V.,Goudet,C.,Nonell,S.,Nicoletti,F.,Llebaria,A.和Ciruela,F。(2017)。 体内疼痛的光学控制用光敏mGlu5受体负变构调节剂 Elife 6.
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引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Notartomaso, S., Scarselli, P., Di Pietro, P., Battaglia, G., Llebaria, A., Ciruela, F. and Nicoletti, F. (2018). Mechanical Allodynia Assessment in a Murine Neuropathic Pain Model. Bio-protocol 8(2): e2671. DOI: 10.21769/BioProtoc.2671.
  2. Font, J., Lopez-Cano, M., Notartomaso, S., Scarselli, P., Di Pietro, P., Bresoli-Obach, R., Battaglia, G., Malhaire, F., Rovira, X., Catena, J., Giraldo, J., Pin, J. P., Fernandez-Duenas, V., Goudet, C., Nonell, S., Nicoletti, F., Llebaria, A. and Ciruela, F. (2017). Optical control of pain in vivo with a photoactive mGlu5 receptor negative allosteric modulator. Elife 6.