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Pain Assessment Using the Rat and Mouse Formalin Tests

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The Journal of Neuroscience
Apr 2014



The formalin test was originally developed by Dubuisson and Dennis (1977), and has since been extensively used to assess pain-related responses. Rats and mice are the most frequently used animal models, though other species including cats, rabbits, guinea pigs, Octodon degus, domestic fowls, crocodiles, tortoises, toads and primates have also been employed. The injection of formalin into the skin of rodent hindpaws to cause spontaneous pain-related flinch behaviors is the most commonly used procedure. The resulting nociceptive response can be divided into two phases differing in timing, duration and underlying mechanisms, and is responsive to many classes of analgesic drugs (Coderre et al., 1990; Hunskaar and Hole, 1987; Rosland et al., 1990; Taylor et al., 1995). The behavioral and electrophysiological responses to formalin consist of an acute phase (Phase I) of a short-lasting response, which is believed to reflect the activity of C-fiber afferent nociceptors. After a short quiescent period, the acute phase is followed by a continuous prolonged response (Phase II), which is believed to be due to central sensitization of the spinal dorsal horn neurons as a result of the initial barrage of input from C-fiber nociceptive afferents during the early phase (Coderre et al., 1993; Dickenson and Sullivan, 1987; Raboisson et al., 1995; Shibata et al., 1989). In this respect, the formalin test has been regarded as a more satisfactory model of pain than tests producing phasic pain like the hot-plate and tail-flick tests (Abbott et al., 1981). Here, we describe the procedures for generating an efficient and reproducible formalin test in rats and mice.

Keywords: Formalin test (福尔马林), Pain (疼痛), Rat (大鼠), Mice (老鼠), Spinal cord (脊髓)

Materials and Reagents

  1. Eight-week-old Wistar (or other strain) rats (body weight 200-250 g)
  2. Eight-week-old Swiss mice or a variety of transgenic/knockout mice (body weight 20-25 g)
  3. Sterile normal saline (0.9% NaCl solution) (Thermo Fisher Scientific, catalog number: BR0053G ) or other vehicles for the test articles
  4. Formaldehyde solution (37 wt.% in H2O) (Sigma-Aldrich, catalog number: 252549 )
  5. 5% formalin in saline (see Recipes)


  1. Body weight scales for rats (range 0.01-2,000 g) (Shenzhen Amput Electronic Technology Co., catalog number: APTP452) and mice (range 0.01-300 g) (Shenzhen Amput Electronic Technology Co., catalog number: APTP457B)
  2. Permanent black marker (Thermo Fisher Scientific, catalog number: S02727)
  3. Individual transparent polycarbonate observation chambers with hinged lids (the formalin test chamber: 30 x 20 x 15 cm for rats and 15 x 15 x 15 cm for mice)
  4. Mirrors (two or more mirrors are recommended to cover the experimental area)
  5. Thick cotton bag with an open end
  6. 0.5-ml syringes with 28-G needles (Thermo Fisher Scientific, catalog number: 22-004-271) or 50-μl Hamilton syringes with 30-G needles (Hamilton Company, catalog number: 80508)
  7. Hand-held counters
  8. Timers (two timers are recommended)

    Figure 1. Example preparation for the rat formalin test


  1. Prism software (version 5.01, GraphPad Software Inc.)


  1. Remove the animal from the home cage and record its body weight. Mark the hindpaw that will be injected with formalin with a permanent black marker.
  2. Place the marked animal into the formalin test chamber that will be used during testing. Place the mirrors behind and beside the container to ensure that the hindpaws can be seen from all angles. It is recommended to have two mirrors used, one is behind the animals and the other one is beside or below the animals. Allow animals to acclimate for 15 to 30 min.
  3. Prepare the 5% formalin solution in saline. For rats, load 50 μl of 5% formalin in a 0.5-ml syringes equipped with a 28-G needle; for mice, load 10 μl of 5% formalin in a 50-μl Hamilton syringes equipped with a 30-G needle. Load one formalin syringe for each animal to be tested.
  4. Remove the animal from its container and place it in the thick cotton bag with the marked hindpaw outside in the open end (the thick cotton bag is used to restrain the animal movement when doing the formalin injection). Inject the 5% formalin solution into the dorsal surface of the paw by placing the needle above the toes and below the ankle and inserting it beneath the surface of the skin.

    Figure 2. Example pictures showing the formalin injection on the rat A and mouse B. a and b showing the syringes used for rat and mouse; c and d showing the injection sites on the rat and mouse, respectively.

  5. Immediately return the injected animal to its container, and start the timer to mark the beginning of Phase I. Flinching of the paw is the most consistent behavioral response in rats after formalin injection. In contrast, mice primarily show paw-licking behaviors.
  6. Repeat formalin injections for the remaining animals. Make every effort to complete injections as quickly as possible. Generally, one investigator can monitor six rats or two mice during a single experiment lot.
    1. For rats, observe the first rat for 1 min and count the number of flinches of the injected hindpaw. Repeat for the remaining five rats. When the sixth rat has been observed, repeat the sequence starting with the first rat in 1-min epochs at 10-min intervals beginning immediately after formalin injection and ending 90 min later. The Phase I response occurs during the first 1 min, and the Phase II response occurs from 20-90 min after formalin injection.
      1. When quantifying flinches, a brisk elevation of the foot is scored as a single flinch. At times, the animal may exhibit several fast flinches that are impossible to quantify. These episodes are typically scored as one count.
    2. For mice, observe for 5 min and record the time spent licking during this period. Inject the next mouse 5 min after the first one and record the licking time. Then observe from 20-40 min after formalin injection.
      1. Like in rats, Phase I and II occur 0-5 min and 20-40 min after the formalin injection, respectively.
      2. It is recommended that prior to running tests with treatments of interest, investigators inject a few control animals to familiarize themselves with the different flinching behaviors that occur during Phase I and II.
    3. Refer to Video 1 for the formalin injection procedures and formalin-induced flinch behaviors in the rat and mouse.

      Video 1. The formalin injection procedures and formalin-induced flinch behaviors in the rat and mouse

  7. Record the total number of flinches in rats or the total time licking in mice for each animal during Phase I and II. Repeat steps 1 to 8 until the desired number of animals (e.g., n = 6-8) in each treatment group have been studied. With each run, it is recommended to have as many treatments as possible presented in parallel.
  8. Perform data analysis to determine statistical significance. For rats, data may be presented as total flinches per minute for Phase I and II (Figure 3A). The area under curve (AUC) over 10-90 minutes may be calculated for Phase II using the Prism software (Gong et al., 2014a; Gong et al., 2011; Gong et al., 2014b; Gong et al., 2014c; Zhang et al., 2013). For mice, data may be presented as the total licking time during 0-5 min for Phase I (the acute phase) and 20-40 min for Phase II (the tonic phase) (Figure 3B) (Gong et al., 2012; Lu et al., 2012; Zhu et al., 2014).

    Figure 3. Typical data of the formalin tests from rats A and mice B. In the rat formalin test, the nociceptive behavior was quantified by counting the number of the formalin-injected paw flinches in 1-min epochs on rats. Subcutaneous injection of formalin in rats produced a characteristic bi-phasic flinching response consisting of an initial, rapidly decaying acute phase (Phase I, within 10 minutes after formalin injection) followed by a slowly rising and long-lived (10-90 min) tonic phase (Phase II). In the mouse, the nociceptive behavior was quantified by counting the total licking time. Subcutaneous injection of formalin in mice produced a characteristic bi-phasic licking response consisting of an initial, rapidly decaying acute phase (Phase I, within 5 minutes after formalin injection) followed by a slowly rising and long-lived (20-40 min) tonic phase (Phase II). Data are presented as means ± SEM (n = 6 in each group). More representative figures have been published elsewhere (Gong et al., 2014a; Gong et al., 2011; Gong et al., 2014b; Gong et al., 2012; Gong et al., 2014c; Lu et al., 2012; Zhang et al., 2013; Zhu et al., 2014).


  1. 5% formalin in saline
    50 μl formaldehyde solution (37 wt. % in H2O)
    950 μl sterile normal saline (0.9% NaCl solution)


This work was supported by the National Natural Science Foundation of China (No. 81374000), the Doctoral Mentor Fund (No. 20110073110062) from the Ministry of Education of China, and Predoctoral Fellowship to N.G. from the Ministry of Education of China, Shanghai Jiao Tong University, and SJTU School of Pharmacy.


  1. Abbott, F. V., Franklin, K. B., Ludwick, R. J. and Melzack, R. (1981). Apparent lack of tolerance in the formalin test suggests different mechanisms for morphine analgesia in different types of pain. Pharmacol Biochem Behav 15(4): 637-640.
  2. Coderre, T. J., Katz, J., Vaccarino, A. L. and Melzack, R. (1993). Contribution of central neuroplasticity to pathological pain: review of clinical and experimental evidence. Pain 52(3): 259-285.
  3. Coderre, T. J., Vaccarino, A. L. and Melzack, R. (1990). Central nervous system plasticity in the tonic pain response to subcutaneous formalin injection. Brain Res 535(1): 155-158.
  4. Dickenson, A. H. and Sullivan, A. F. (1987). Peripheral origins and central modulation of subcutaneous formalin-induced activity of rat dorsal horn neurones. Neurosci Lett 83(1-2): 207-211.
  5.  Dubuisson, D. and Dennis, S. G. (1977). The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats. Pain 4(2): 161-174.
  6. Gong, N., Fan, H., Ma, A. N., Xiao, Q. and Wang, Y. X. (2014a). Geniposide and its iridoid analogs exhibit antinociception by acting at the spinal GLP-1 receptors. Neuropharmacology 84: 31-45.
  7. Gong, N., Gao, Z. Y., Wang, Y. C., Li, X. Y., Huang, J. L., Hashimoto, K. and Wang, Y. X. (2011). A series of D-amino acid oxidase inhibitors specifically prevents and reverses formalin-induced tonic pain in rats. J Pharmacol Exp Ther 336(1): 282-293.
  8. Gong, N., Li, X. Y., Xiao, Q. and Wang, Y. X. (2014b). Identification of a novel spinal dorsal horn astroglial D-amino acid oxidase-hydrogen peroxide pathway involved in morphine antinociceptive tolerance. Anesthesiology 120(4): 962-975.
  9. Gong, N., Wang, Y. C., Wang, H. L., Ma, A. N., Hashimoto, K. and Wang, Y. X. (2012). Interactions of the potent D-amino acid oxidase inhibitor CBIO with morphine in pain and tolerance to analgesia. Neuropharmacology 63(3): 460-468.
  10. Gong, N., Xiao, Q., Zhu, B., Zhang, C. Y., Wang, Y. C., Fan, H., Ma, A. N. and Wang, Y. X. (2014b). Activation of spinal glucagon-like peptide-1 receptors specifically suppresses pain hypersensitivity. J Neurosci 34(15): 5322-5334.
  11. Hunskaar, S. and Hole, K. (1987). The formalin test in mice: dissociation between inflammatory and non-inflammatory pain. Pain 30(1): 103-114.
  12. Lu, J. M., Gong, N., Wang, Y. C. and Wang, Y. X. (2012). D-Amino acid oxidase-mediated increase in spinal hydrogen peroxide is mainly responsible for formalin-induced tonic pain. Br J Pharmacol 165(6): 1941-1955.
  13. Raboisson, P., Dallel, R., Clavelou, P., Sessle, B. J. and Woda, A. (1995). Effects of subcutaneous formalin on the activity of trigeminal brain stem nociceptive neurones in the rat. J Neurophysiol 73(2): 496-505.
  14. Rosland, J. H., Tjolsen, A., Maehle, B. and Hole, K. (1990). The formalin test in mice: effect of formalin concentration. Pain 42(2): 235-242.
  15. Shibata, M., Ohkubo, T., Takahashi, H. and Inoki, R. (1989). Modified formalin test: characteristic biphasic pain response. Pain 38(3): 347-352.
  16. Taylor, B. K., Peterson, M. A. and Basbaum, A. I. (1995). Persistent cardiovascular and behavioral nociceptive responses to subcutaneous formalin require peripheral nerve input. J Neurosci 15(11): 7575-7584.
  17. Zhang, J. Y., Gong, N., Huang, J. L., Guo, L. C. and Wang, Y. X. (2013). Gelsemine, a principal alkaloid from Gelsemium sempervirens Ait., exhibits potent and specific antinociception in chronic pain by acting at spinal alpha3 glycine receptors. Pain 154(11): 2452-2462.
  18. Zhu, B., Gong, N., Fan, H., Peng, C. S., Ding, X. J., Jiang, Y. and Wang, Y. X. (2014c). Lamiophlomis rotata, an orally available tibetan herbal painkiller, specifically reduces pain hypersensitivity states through the activation of spinal glucagon-like peptide-1 receptors. Anesthesiology 121(4): 835-851.


福尔马林试验最初由Dubuisson和Dennis(1977)开发,此后被广泛用于评估疼痛相关的反应。大鼠和小鼠是最常用的动物模型,但其他物种,包括猫,兔,豚鼠,八达冬,家禽,鳄鱼,龟,蟾蜍和灵长目动物也被使用。将福尔马林注射到啮齿动物后爪皮肤中引起自发性疼痛相关的退缩行为是最常用的手术。所产生的伤害感受反应可以分为时间,持续时间和潜在机制不同的两个阶段,并且对许多类别的止痛药有反应(Coderre等,1990; Hunskaar和Hole,1987; Rosland等,1990; Taylor et al。,1995)。对福尔马林的行为和电生理反应由短期持续反应的急性期(I期)组成,据信其反映C纤维传入伤害感受器的活性。在短暂的静止期后,急性期之后是连续延长的反应(II期),这被认为是由于脊髓背角神经元的中枢致敏引起的,因为来自C纤维伤害感受器的输入的初始阻塞早期阶段的传入(Coderre等,1993; Dickenson和Sullivan,1987; Raboisson等,1995; Shibata等,1989)。在这方面,福尔马林试验被认为是一种更加令人满意的疼痛模型,而不是产生诸如热板和尾部轻弹试验之类的阶段性疼痛的试验(Abbott等,1981)。在这里,我们描述了在大鼠和小鼠中产生有效和可重复的福尔马林试验的程序。

关键字:福尔马林, 疼痛, 大鼠, 老鼠, 脊髓


  1. 八周大的Wistar(或其他应变)大鼠(体重200-250g)
  2. 8周龄的Swiss小鼠或各种转基因/敲除小鼠(体重20-25g)
  3. 无菌生理盐水(0.9%NaCl溶液)(Thermo Fisher Scientific,目录号:BR0053G)或用于测试物品的其他载体
  4. 甲醛溶液(37重量%的H 2 O)(Sigma-Aldrich,目录号:252549)
  5. 5%福尔马林的盐水溶液(见配方)


  1. 体重(体重0.01-2000克)(深圳市安普电子科技有限公司,目录号:APTP452)和老鼠(范围0.01-300克)(深圳市安普电子科技有限公司,目录号:APTP457B) >
  2. 永久黑标记(Thermo Fisher Scientific,目录号:S02727)
  3. 具有铰链盖的单个透明聚碳酸酯观察室(福尔马林试验室:大鼠为30×20×15cm,小鼠为15×15×15cm)
  4. 镜子(建议两个或多个镜子覆盖实验区域)
  5. 厚棉袋,开口端
  6. 具有28-G针(Thermo Fisher Scientific,目录号:22-004-271)或50-μl具有30-G针的Hamilton注射器(Hamilton Company,目录号:80508)的0.5ml注射器
  7. 手持式计数器
  8. 计时器(推荐两个计时器)



  1. Prism软件(版本5.01,GraphPad Software Inc.)


  1. 从家笼中取出动物,并记录其体重。 标记将用福尔马林注射永久性黑色标记的后爪。
  2. 将标记的动物放入福尔马林测试室,将在测试期间使用。 将镜子放在容器后面和侧面,以确保从所有角度都能看到后爪。 建议使用两个镜子,一个在动物后面,另一个在动物旁边或下面。 允许动物适应15至30分钟。
  3. 制备盐水中的5%福尔马林溶液。 对于大鼠,在装有28-G针的0.5-ml注射器中加载50μl的5%福尔马林; 对于小鼠,在装有30-G针的50-μlHamilton注射器中装载10μl5%福尔马林。 为每个待测试的动物装上一个福尔马林注射器。
  4. 从其容器中取出动物,并将其放在厚棉布袋中,标记的后爪外侧在开口端(厚的棉袋用于限制动物运动时,福尔马林注射)。将5%福尔马林溶液注入爪子的背面,将针头放在脚趾上方,脚踝下方,将其插入皮肤表面下方。

    图2.显示对大鼠A和小鼠B的福尔马林注射的实施例照片 a和b显示用于大鼠和小鼠的注射器; c和d分别表示大鼠和小鼠的注射部位
  5. 立即将注射的动物返回到其容器中,并启动计时器以标记I期的开始。在福尔马林注射后,爪的退缩是大鼠中最一致的行为反应。相反,小鼠主要显示爪舔行为
  6. 重复福尔马林注射剩余的动物。尽一切努力尽快完成注射。通常,一个研究者可以在单个实验批中监测六只大鼠或两只小鼠。
    1. 对于大鼠,观察第一只大鼠1分钟并计数 注射后爪的退缩。对剩余的5只大鼠重复。 当观察到第六只大鼠时,重复从开始的序列 第一只大鼠在1分钟的时间间隔以10分钟的间隔立即开始 福尔马林注射后90分钟后结束。 第一阶段反应 发生在前1分钟,而II期反应发生于 在福尔马林注射后20-90分钟。
      1. 当量化衰退时,a 脚的轻快高度被评分为单个退缩。 有时, 动物可能表现出几个不可能的快速衰退 量化。 这些情节通常被计为一个计数。
    2. 对于 小鼠,观察5分钟,并记录在此期间舔舔所花费的时间 期。 注射下一个鼠标5分钟后第一个和记录 舔时间。 然后在20-40分钟后观察福尔马林注射。
      1. 与大鼠一样,I期和II期分别在注射福尔马林后0-5分钟和20-40分钟出现
      2. 建议在运行测试之前使用 兴趣,调查人员注射几种对照动物熟悉 本身与在其间发生的不同的退缩行为 阶段I和II。
    3. 参考视频1的福尔马林注射程序和福尔马林诱导的大鼠和老鼠的退缩行为。

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  7. 记录在I期和II期每只动物的大鼠退缩总数或小鼠总舔时间。重复步骤1至8,直到研究了每个治疗组中所需数量的动物(例如,n = 6-8)。每次运行时,建议尽可能多地平行处理。
  8. 执行数据分析以确定统计显着性。对于大鼠,数据可以表示为阶段I和II的每分钟总衰退(图3A)。可以使用Prism软件(Gong等人,2014a; Gong等人,2011年),计算10-90分钟内曲线下面积(AUC) ; Gong等人,2014b; Gong等人,2014c; Zhang等人,2013)。对于小鼠,数据可以表示为I期(急性期)0-5分钟期间的总舔食时间和II期(强直期)(图3B)20-40分钟的总舔食时间(Gong等, ,2012; Lu 等人,2012; Zhu 等人,2014)。

    图3.来自大鼠A和小鼠B的福尔马林试验的典型数据在大鼠福尔马林试验中,通过计数在1分钟时期内注射福尔马林的爪爪的数量来定量伤害行为对大鼠。在大鼠中皮下注射福尔马林产生特征性的双阶段急性反应,其由初始的,快速衰变的急性期(I期,在福尔马林注射后10分钟内),随后缓慢上升和长期(10-90min)相(II期)。在小鼠中,通过计数总舔时间来定量伤害行为。皮下注射福尔马林在小鼠中产生特征性的两阶段舔反应,其由初始的快速衰变的急性期(I期,在福尔马林注射后5分钟内),随后缓慢上升和长期(20-40分钟)强直相(II期)。数据表示为平均值±SEM(每组n = 6)。更具代表性的数字已经在其他地方公开(Gong等人,2014a; Gong等人,2011; Gong等人,2014b; Gong等人,2012; Gong等人,2014c; Lu等人,2012; Zhang等人,2012;/em>,2013; Zhu ,,2014)


  1. 5%福尔马林的盐水溶液
    50μl甲醛溶液(37重量%在H 2 O中)


这项工作得到了中国国家自然科学基金(No.81374000),中国教育部博士生导师基金(No. 20110073110062)和中国教育部授予前授予奖学金。 来自中国教育部,上海交通大学和中国药科大学。


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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. Gong, N., Huang, Q., Chen, Y., Xu, M., Ma, S. and Wang, Y. (2014). Pain Assessment Using the Rat and Mouse Formalin Tests. Bio-protocol 4(21): e1288. DOI: 10.21769/BioProtoc.1288.
  2. Gong, N., Xiao, Q., Zhu, B., Zhang, C. Y., Wang, Y. C., Fan, H., Ma, A. N. and Wang, Y. X. (2014b). Activation of spinal glucagon-like peptide-1 receptors specifically suppresses pain hypersensitivity. J Neurosci 34(15): 5322-5334.