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Assessment of Mechanical Allodynia in Rats Using the Electronic Von Frey Test
使用电子Von Frey测痛仪评估大鼠的机械痛觉超敏

Jeremy FerrierJeremy Ferrier*Fabien MarchandFabien Marchand*David BalayssacDavid Balayssac  (*contributed equally to this work)
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Abstract

Chronic pain is one of the most debilitating conditions, affecting one out of five people worldwide. Preclinical models in rodents represent a valuable tool for the study of pathophysiological mechanisms and the discovery of new analgesic drugs. However, pain evaluation in rodents is rather challenging. Altered response to mechanical or thermal stimuli is commonly used as behavioral outcome to measure pain sensitivity. This protocol introduces a method for assessing static mechanical pain hypersensitivity in rats using an electronic von Frey (VF) test. The electronic VF is an evolution of the manual VF hairs previously described by Kim and Chung (1991). In this previous procedure, 6 calibrated nylon filaments (diameters of 0.13, 0.23, 0.31, 0.48, 0.52, and 0.59 mm, respectively) are perpendicularly applied to the plantar surface of the rat hind paw, to deliver the following defined pressures: 5.89, 9.81, 27.0, 74.4, 124, and 205 mN. Each application has to be repeated several times for each filament to determine the mechanical threshold. Comparatively, the electronic VF is relatively easy-to-use and particularly suited for pharmacological studies with precise time-points. The electronic VF can be used as a behavioural read-out for a wide range of models, including inflammatory and neuropathic pain. The following protocol was originally published in Ferrier et al. (2013), Grégoire et al. (2014) and in Ferrier et al. (2015).

Keywords: Electronic von frey(电子Von Frey), Allodynia(异常性疼痛), Rat(老鼠), Neuropathy(神经病), Pain(疼痛)

Materials and Reagents

  1. Plastic tip (Harvard Apparatus, catalog number: 76-0488 )
    Note: The tip doesn’t need to be changed between rats and between sessions as long as it is not damaged or dirty beyond cleaning.
  2. Rats
    Note: To reach a sufficient statistical power, use a minimum of 8 rats per group. Use preferentially Sprague-Dawley male rats, weighing 200 to 400 g (6 to 12 weeks old), since this protocol has only been validated on this strain of rats. However, there are no obvious reasons that this protocol won’t be suitable for others strains of rats.
  3. Cleaning solution
    Note: Within one session, the boxes are carefully cleaned with water to remove the odor of the previous animal. At the end of the session, clean the boxes using 70% ethanol.
  4. Oxaliplatin (6 mg/kg, dissolved in 5% glucose)

Equipment

  1. Apparatus (either home-made or commercially available at Harvard Apparatus, catalog number: 76-0886/87 )
    Note: The apparatus consists of an elevated horizontal wire mesh stand (Figure 1). The dimensions of the Plexiglas boxes set on top of the wire grid are 17 x 11 x 13 cm. The boxes are covered with perforated lids. Frosted or opaque separators can be used to diminish interactions between rats.


    Figure 1. Apparatus for the electronic von Frey test

  2. Electronic von Frey test (BIO-EVF4, Bioseb®, France)
    Note: The apparatus consists of a portable force transducer fitted with a plastic tip (Figure 2). When applying a pressure on the tip, the maximal force applied (in grams) is automatically recorded by the electronic device and displayed on the screen. The maximal pressure (cut-off) will be reached when the paw of the animal is lifted by the plastic tip. Between two stimulations (separated by at least 3 min), the force value is set to zero simply by pushing a foot pedal.


    Figure 2. Electronic von Frey test device

Procedure

Notes:
a. Local animal ethics/protocol regarding animal housing and handling need to be followed.
b. The electronic VF test measures the animal’s mechanical “pain” hypersensitivity, a parameter which can be influenced by stress/anxiety (Gamaro et al., 1998). Therefore, it is very important to minimize any environmental stimuli (visual or auditory) and allow rats to acclimate to the experimental room, the test and the experimenter. It should be stressed out that experiments need to be performed in a quiet room by a blinded experimenter using the method of equal blocks with randomisation of treatments to avoid any uncontrollable environmental influence that might induce a modification in the behavioural response.
c. If rats have not been subjected to any other test, put their home-cages in the experimental room for at least one hour for a few days before the experiments. Meanwhile, habituate the animals to be gently held for a few seconds to avoid stress related to handling.

  1. Days 1 and 2. Habituation
    1. Rats are transported to the experimental room and stay at least 1 h in their home cages.
    2. Then the first batch of rats is placed on the apparatus for 30 min to acclimatize to the test. With our homemade apparatus, up to 6 rats can be tested simultaneously. Bigger batch can be used depending on study design.
    3. Place a heavy object on the boxes’ lids to prevent rats from escaping.
    4. Clean the boxes with water humidified paper and place the following batch of rats.
    5. This procedure is carried out for two days.

  2. Day 0. Testing
    1. Similar to Days 1 and 2, rats are transported to the experimental room 1 h before testing.
    2. The first batch of rats is placed on the boxes for 15 min.
    3. Place a clean plastic tip on the probe and zero the read-out.
    4. Apply the tip of the force transducer on the middle of the rat hind paw (footpad center) from below (Figure 3). The testing area may depend on the pain model used.


      Figure 3. Application of the von Frey tip on the rat’s hind paw

    5. Important step: Increase the pressure gradually (approximately 15 g/sec) and linearly until a clear withdrawal of the paw is observed.
    6. Note the pressure value that elicited the reaction.
    7. Assess the withdrawal threshold on the rat situated in the adjacent box (there is no need to replace the plastic tip between each animal). Repeat steps B4-7 until each rat has been submitted to the test. Then go back to the first rat and repeat until two close withdrawal thresholds are observed (less than 10% of difference). Stimulations on the same hind paw should be at least 3-5 min apart.
    8. Both hind paw can be assessed in the same animal, but in the case of a polyneuropathy (Ferrier et al., 2015), there is no need to test both of them. In the case of mononeuropathy [e.g., spinal nerve injury (Kim and Chung, 1992) or chronic constriction injury (Bennett and Xie, 1988)], the paw withdrawal threshold of the contralateral limb (unaffected) can serve as an intra-animal control.

Representative data



Figure 4. Representative results. Paw withdrawal thresholds were measured using the electronic von Frey test before and after intraperitoneal administration of oxaliplatin (6 mg/kg, dissolved in 5% glucose) or its vehicle at Day 0. Rats treated with oxaliplatin developed a transient mechanical allodynia from Day 2 to Day 4 after the administration (***P < 0.001 compared to vehicle-treated rats, two-way repeated measure ANOVA followed by a Bonferroni post hoc test) as described in (Ferrier et al., 2013). Data are represented as mean ± standard deviation.

Notes

  1. The rats have to hold still during the procedure with their entire hind paw on the floor. The measurement should be excluded if the rat has moved during the stimulation.
  2. The first value recorded is often excluded from the analysis, as rats may be surprised by the stimulation and reacted before reaching pain thresholds. Therefore, a minimum of 3 measurements are taken for each rat. Both hind paws can be tested per animal.
  3. Make sure to apply the tip perpendicularly to the hind paw and with the same speed between rats as it may increase variability of the results.
  4. If rats are tested too many times over several weeks, they may become unresponsive to the test or fall asleep in the boxes. Very gently tapping or running a pen along the wire grid before the stimulation (but not during) may help increase attention and improve the results.
  5. Healthy rats yield paw withdrawal thresholds between 60 to 80 g. This value may be directly dependent upon the experimenter, especially regarding the speed of which the tip is applied on the hind paw (slowly: low thresholds and quickly: high thresholds).

Acknowledgments

This work was funded by the Ligue Nationale Contre le Cancer comité du Puy de Dome.

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. Ferrier, J., Bayet-Robert, M., Dalmann, R., El Guerrab, A., Aissouni, Y., Graveron-Demilly, D., Chalus, M., Pinguet, J., Eschalier, A., Richard, D., Daulhac, L., Marchand, F. and Balayssac, D. (2015). Cholinergic neurotransmission in the posterior insular cortex is altered in preclinical models of neuropathic pain: key role of muscarinic M2 receptors in donepezil-induced antinociception. J Neurosci 35(50): 16418-16430.
  3. Ferrier, J., Bayet-Robert, M., Pereira, B., Daulhac, L., Eschalier, A., Pezet, D., Moulinoux, J. P. and Balayssac, D. (2013). A polyamine-deficient diet prevents oxaliplatin-induced acute cold and mechanical hypersensitivity in rats. PLoS One 8(10): e77828.
  4. Gamaro, G. D., Xavier, M. H., Denardin, J. D., Pilger, J. A., Ely, D. R., Ferreira, M. B. and Dalmaz, C. (1998). The effects of acute and repeated restraint stress on the nociceptive response in rats. Physiol Behav 63(4): 693-697.
  5. Gregoire, S., Wattiez, A. S., Etienne, M., Marchand, F. and Ardid, D. (2014). Monoarthritis-induced emotional and cognitive impairments in rats are sensitive to low systemic doses or intra-amygdala injections of morphine. Eur J Pharmacol 735: 1-9.
  6. Kim, S. H. and Chung, J. M. (1991). Sympathectomy alleviates mechanical allodynia in an experimental animal model for neuropathy in the rat. Neurosci Lett 134(1): 131-134.
  7. 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.

简介

慢性疼痛是最恶化的疾病之一,影响全球五分之一的人。啮齿动物的临床前模型是研究病理生理机制和发现新的镇痛药物的有价值的工具。然而,啮齿动物的疼痛评估是相当有挑战性的。对机械或热刺激的改变反应通常用作测量疼痛敏感性的行为结果。该方案介绍了使用电子冯Frey(VF)测试评估大鼠静态机械性疼痛超敏反应的方法。电子VF是Kim和Chung(1991)先前描述的手动VF毛发的演变。在以前的程序中,将6根经校准的尼龙细丝(直径分别为0.13,0.23,0.31,0.48,0.52和0.59mm)垂直施加到大鼠后爪的足底表面,以提供以下规定的压力:5.89, 9.81,27.0,74.4,124和205 mN。每个应用必须对每个灯丝重复几次以确定机械阈值。相比之下,电子VF相对易于使用,特别适用于具有精确时间点的药理学研究。电子VF可用作各种模型的行为读出,包括炎症和神经性疼痛。以下协议最初在Ferrier等人发表。 (2013),Grégoireet al。 (2014)和Ferrier等(2015年)。

关键字:电子Von Frey, 异常性疼痛, 老鼠, 神经病, 疼痛

材料和试剂

  1. 塑料尖端(Harvard Apparatus,目录号:76-0488)
    注意:只要没有损坏或肮脏,清洁后,老鼠之间和会话之间不需要更换提示。
  2. 大鼠
    注意:为了达到足够的统计效力,每组至少使用8只大鼠。 优先使用体重200至400克(6至12周龄)的Sprague-Dawley雄性大鼠,因为该方案仅在该大鼠品系上验证。 然而,没有明显的理由,此协议将不适合其他品系的老鼠。
  3. 清洁溶液
    注意:在一个会话中,小心地用水小心清洁以去除以前的动物的气味。 在会话结束时,使用70%乙醇清洁盒子。
  4. 奥沙利铂(6mg/kg,溶于5%葡萄糖)

    材料和试剂

    1. 塑料尖端(Harvard Apparatus,目录号:76-0488)
      注意:只要没有损坏或肮脏,清洁后,老鼠之间和会话之间不需要更换提示。
    2. 大鼠
      注意:为了达到足够的统计效力,每组至少使用8只大鼠。 优先使用体重200至400克(6至12周龄)的Sprague-Dawley雄性大鼠,因为该方案仅在该大鼠品系上验证。 然而,没有明显的理由,此协议将不适合其他品系的老鼠。
    3. 清洁溶液
      注意:在一个会话中,小心地用水小心清洁以去除以前的动物的气味。 在会话结束时,使用70%乙醇清洁盒子。
    4. 奥沙利铂(6mg/kg,溶于5%葡萄糖)
      ...

      材料和试剂

      1. 塑料尖端(Harvard Apparatus,目录号:76-0488)
        注意:只要没有损坏或肮脏,清洁后,老鼠之间和会话之间不需要更换提示。
      2. 大鼠
        注意:为了达到足够的统计效力,每组至少使用8只大鼠。 优先使用体重200至400克(6至12周龄)的Sprague-Dawley雄性大鼠,因为该方案仅在该大鼠品系上验证。 然而,没有明显的理由,此协议将不适合其他品系的老鼠。
      3. 清洁溶液
        注意:在一个会话中,小心地用水小心清洁以去除以前的动物的气味。 在会话结束时,使用70%乙醇清洁盒子。
      4. 奥沙利铂(6mg/kg,溶于5%葡萄糖)
        ......
      5. 第1天和第2天。习惯
        1. 大鼠被运到实验室,在他们的笼子里至少保持1小时
        2. 然后将第一批大鼠置于该装置上30分钟以适应该试验。 用我们的自制装置,可同时测试多达6只大鼠。 根据研究设计,可以使用更大的批次。
        3. 将重物放在盒子的盖子上,以防止老鼠逃跑。
        4. 用水加湿纸清洁盒子,并放置以下批次的大鼠。
        5. 此程序执行两天。

      6. 测试
        1. 类似于第1天和第2天,大鼠在测试前1小时被运送到实验室
        2. 将第一批大鼠置于盒子上15分钟。
        3. 将一个干净的塑料尖端放在探针上,并将读数归零。
        4. 将力传感器的尖端应用于大鼠后爪(足垫中心)的中部从下面(图3)。 测试区域可能取决于所使用的疼痛模型

          图3. von Frey吸头在大鼠后爪上的应用

        5. 重要步骤:逐渐增加压力(约15g/sec),直线直到观察到爪子清晰退出
        6. 注意引起反应的压力值。
        7. 评估位于相邻盒子中的大鼠的撤回阈值(不需要替换每只动物之间的塑料尖端)。重复步骤B4-7,直到每只大鼠进行测试。然后回到第一只大鼠,并重复,直到观察到两个收缩阈值(小于差异的10%)。同一后爪上的刺激应至少间隔3-5分钟
        8. 两个后爪可以在相同的动物中评估,但是在多发性神经病的情况下(Ferrier等人,2015),不需要测试它们两者。在单神经病例如脊髓神经损伤(Kim和Chung,1992)或慢性压迫性损伤(Bennett和Xie,1988)的情况下,对侧肢(未受影响的)的缩足阈值,可作为动物内控制

      代表数据



      图4.代表性结果。在第0天腹膜内施用奥沙利铂(6mg/kg,溶解于5%葡萄糖)或其载体之前和之后,使用电子von Frey试验测量爪撤回阈值。用奥沙利铂治疗的大鼠在给药后从第2天到第4天发生瞬时机械性异常性疼痛(与媒介物治疗的大鼠相比,*** P 0.001,双向重复测量ANOVA,随后是Bonferroni事后检验),如(Ferrier等人,2013)中所述。数据表示为平均值±标准偏差。

      笔记

      1. 大鼠在手术期间必须保持静止,其整个后爪在地板上。如果大鼠在刺激期间移动,则应排除测量。
      2. 记录的第一个值通常从分析中排除,因为大鼠可以通过刺激而惊讶并在达到疼痛阈值之前反应。因此,对每只大鼠进行最少3次测量。两只后爪可以每只动物进行测试。
      3. 确保将尖端垂直地应用于后爪,并在大鼠之间以相同的速度,因为它可能增加结果的变异性。
      4. 如果大鼠在几周内测试太多次,它们可能对测试无反应或在盒子中睡着。在刺激(但不是刺激)之前,轻轻地沿着线网轻轻敲击或跑动笔可能有助于提高注意力并改善结果。
      5. 健康大鼠产生60至80g的缩足阈值。该值可以直接取决于实验者,特别是关于尖端施加在后爪上的速度(缓慢地:低阈值和快速:高阈值)。

      致谢

      这项工作由Ligue Nationale Contrele癌症联合会de Puy de Dome资助。

      参考文献

      1. Bennett,GJ和Xie,YK(1988)。  A外周单神经病在大鼠中产生像人类中所见的疼痛感觉的病症。疼痛 33(1):87-107。
      2. Ferrier,J.,Bayet-Robert,M.,Dalmann,R.,El Guerrab,A.,Aissouni,Y.,Graveron-Demilly,D.,Chalus,M.,Pinguet,J.,Eschalier, Richard,D.,Daulhac,L.,Marchand,F.和Balayssac,D。(2015)。  在后岛地皮质中的胆碱能神经传递在神经性疼痛的临床前模型中改变:毒蕈碱M2受体在多奈哌齐诱导的抗感受伤害中的关键作用。 em> 35(50):16418-16430。
      3. Ferrier,J.,Bayet-Robert,M.,Pereira,B.,Daulhac,L.,Eschalier,A.,Pezet,D.,Moulinoux,JP和Balayssac,D。(2013) "多胺缺乏的饮食可以预防大鼠中奥沙利铂引起的急性感冒和机械性超敏反应。"


        /a> PLoS One 8(10):e77828。
      4. Gamaro,GD,Xavier,MH,Denardin,JD,Pilger,JA,Ely,DR,Ferreira,MB和Dalmaz,C。(1998)。  交感神经切除术减轻了大鼠神经病变的实验动物模型中的机械性异常性疼痛。 Neurosci Lett 134(1):131-134。
      5. Kim,SH and Chung,JM(1992)。  An实验模型用于在大鼠中通过节段性脊神经结扎产生的周围神经病变。 50(3):355-363。
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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. Ferrier, J., Marchand, F. and Balayssac, D. (2016). Assessment of Mechanical Allodynia in Rats Using the Electronic Von Frey Test. Bio-protocol 6(18): e1933. DOI: 10.21769/BioProtoc.1933.
  2. Ferrier, J., Bayet-Robert, M., Dalmann, R., El Guerrab, A., Aissouni, Y., Graveron-Demilly, D., Chalus, M., Pinguet, J., Eschalier, A., Richard, D., Daulhac, L., Marchand, F. and Balayssac, D. (2015). Cholinergic neurotransmission in the posterior insular cortex is altered in preclinical models of neuropathic pain: key role of muscarinic M2 receptors in donepezil-induced antinociception. J Neurosci 35(50): 16418-16430.
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