Formalin Murine Model of Pain

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Apr 2017



Pain research is mostly based on experimental assays that use animal models, which may allow deciphering the physiopathology of this condition and to propel drug discovery. The formalin nociception test is considered one of the most predictive approaches to study acute pain in rodents. This test permits monitoring pain-related responses (i.e., itch) caused by a subcutaneous injection of an inflammatory agent, namely 2.5% formalin solution, in the hind paw. After the injection, two distinct time periods or phases of licking/biting behaviour occur, which are separated by a quiescent period. Importantly, these phases differ in duration and underlying mechanisms. Hence, the initial acute phase (phase I), commonly recorded for 5 min just after formalin administration, reflects acute peripheral pain, probably due to direct activation of nociceptors through TRPA1 channels. On the other hand, the phase II, which starts after the quiescent period (5-15 min) and is commonly recorded for 15-30 min, is due to the ongoing inflammatory input and central nociceptive sensitization. Here, we describe in detail the protocol used to perform a reliable and reproducible formalin test in mice.

Keywords: Pain (疼痛), Nociception (伤害感受), Inflammation (炎症), Analgesia (镇痛), Mice (小鼠)


The formalin test is an experimental assay that permits determining mice nocifensive behaviour. Thus, the mice response (i.e., licking and flinching) is assessed after a subcutaneous injection of formalin normally in the plantar hind paw (Tjolsen et al., 1992). The test was originally described in the late seventies (Dubuisson and Dennis, 1977), and, initially, it consisted of the injection of 50 µl of 5% formalin in the dorsal surface of one forepaw of a rat or a cat. Since then, the formalin test has been extensively used to assess nociception and inflammation-related responses, thus being adapted according to each study’s aim. Mice and rats are frequently used due to their innate grooming behaviour (i.e., forepaw licking). Accordingly, a 2.5% formalin (in physiological saline solution) injected in the mid-plantar surface of the rodent hind paw is commonly used to induce nociceptive responses (i.e., paw ‘flinching’ and licking) lasting for 45-90 min (Tjolsen et al., 1992).

The formalin test is considered a very powerful tool in preclinical research to develop novel analgesic drugs (Mogil, 2009). The main advantage of the test consists of the very objective quantification of the pain-associated behaviour in response to the noxious stimuli (i.e., formalin). Furthermore, the formalin test allows the study of two different kinds of pain typologies: i) acute peripheral pain mediated by the direct activation of nociceptors through TRPA1 channels, and ii) inflammatory and central nociceptive sensitization. Indeed, the existence of these two phases lead to the possibility of studying different analgesic drugs, including NSAIDs and other mild analgesics, and, therefore, may allow deciphering the drug mechanism of action along the pain neuraxis.

Overall, the formalin test may be considered an easy-going experimental approach to ascertain the analgesic activity of any named drug. Here, we provide a complete description of the formalin test in order to facilitate its implementation by other scientists.

Materials and Reagents

  1. Syringes (BD Micro-FineTM Demi, U-100 Insulin, 30 G x ½’’–0.33 x 8 mm) (BD, catalog number: 324826 ) (Figure 1d)
  2. Eppendorf tubes (Figure 1b)
  3. Animals: Adult male CD-1 mice (Charles River Laboratories, L’Arbresle, France; RRID: MGI: 3785721) with 8 weeks of age and weighing 20-25 g were used
    Note: Animals were housed in standard cages with free access to food and water, and were maintained under controlled standard conditions (12 h dark/light cycles starting at 7:30 AM, 22 °C temperature, and 66% humidity). All manipulations were carried out between 9:00 and 16:00 h. Procedures in this study 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 relevant institutions (CEUA/UFSC and CEEA/UB) approved the protocol. Efforts were made to minimize suffering and reduce the number of animals used in the experiments. To reach statistical significance, at least 10 mice per group are highly recommended.
  4. 70% ethanol
  5. Formalin (A 36.5-38% formaldehyde solution in H2O) (Sigma-Aldrich, catalog number: F8775-500ML )
    Note: It was used to prepare the final 2.5% formalin in physiological saline solution.
  6. 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).
  7. Vehicle. Sterile physiological saline (0.9% NaCl) solution (Grifols, catalog number: 605137 )
  8. Sterile cleaning solution
    Note: A 70% ethanol in H2O was used.
  9. 2.5% formalin solution (see Recipes)


  1. Observational chamber
    Circular (25 cm ø x 30 cm height) glass container (i.e., glass beaker) (Figure 1a). The glass container should be cleaned with 70% ethanol first and then with sterile water to remove any animal/ethanol odour. Air dry for 5 min before testing another animal. No access to food or water should be allowed
    Note: A recording system (i.e., camera) could be optionally used. Thus, place the recording system behind, below and/or above the observational chamber. Ensure that the hind paw of the animal is clearly visible from all angles of observation, thus a set of mirrors surrounding the observational chamber could be used for this purpose.
  2. Weighing machine (Figure 1c)
  3. Coloured surface (Figure 1e)
  4. Restrain apparatus for mice (Figure 1f)
    Note: Alternatively, a 50-ml Falcon tube could also be used.
  5. PIPETMAN ClassicTM Pipets (Gilson, model: P20, P200, and P1000, catalog numbers: F123600 , F123601 and F123602 , respectively)
  6. Timer (Figure 1g) and chronometer (Figure 1h)
  7. Personal protective equipment (Figure 1i) (Laboratory coat, gloves, masks, etc.)

    Figure 1. Material needed for the formalin test. Glass container (a), freshly prepared 2.5% formalin solution (b), balance (c), insulin syringe (d), black plastic surface (e), restrain apparatus for mice (f), timer (g), chronometer (h) and gloves (i).


  1. Habituation
    One day before the experiment, the animals to be tested are individually exposed to the observational chamber (Figure 1a) for 1 h.
  2. Reagents preparation
    The day of the experiment, prepare the 2.5% formalin solution (see Recipes) (Figure 1b) and the drug to be tested (not shown). Clean all the material needed.
  3. Weighing animals
    Remove the mouse from the house cage and measure its body weight using a weighing machine (Figure 1c).
  4. Administration of the analgesic drug
    Administer (i.e., intraperitoneally, i.p.) the analgesic drug to be tested (i.e., raseglurant; Figure 2) or the same volume of vehicle (physiological saline solution) using an insulin syringe (Figure 1d).
    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.
  5. Acclimation
    The animal is placed in the observational chamber for 20 min before recording formalin administration (Figure 2).
    Note: A coloured plastic surface (i.e., black) could be placed below the observational chamber for contrasting the animal’s hair (i.e., CD-1 mice have white hair) (Figure 1e).

    Figure 2. Treatment schedule depicting the administration regimen of drug, formalin and the behavioural testing (phase I and II)

  6. Formalin loading
    Load 20 µl of 2.5% formalin solution directly in the 0.3 ml insulin syringe equipped with a 30 G needle.
    Note: Optionally, the 20 µl of 2.5% formalin solution could be taken with a 2-20 µl micropipette, placed in a 100 µl Eppendorf tube and then load the insulin syringe with.
  7. Formalin administration
    Take the animal from the observational cage and place it carefully inside the restrain module (Figure 1f) with the selected (i.e., right) hind paw exposed outside (Figure 3A). Inject slowly the 20 µl of 2.5% formalin solution using the insulin syringe in the mid-plantar surface of the hind paw (Figure 3B).
    1. The needle should be placed in between toes and ankle and inserted beneath the surface of the skin, thus any tissue damage should be avoided.
    2. The injection should be done firmly and time-optimized. Animals could perceive the restrain as stressor stimulus and that can increase the variability of the results.

      Figure 3. Hind paw of a restrained mouse (A) and administration of formalin solution (B)

  8. Phase I (0-5 min)
    Return the injected animal to its observational chamber. Set the timer for 5 min and start it to mark the beginning of the phase I (Figure 2). Use a chronometer to visually measure the total time the animal spends licking or biting the injected paw during this period (5 min) (Video 1). Annotate the result.
    1. If a recording system is used, switch on and off the system accordingly.
    2. We strongly recommend the investigator to perform some training at this step to familiarize themselves with the licking/biting behavior.
  9. Quiescent period (5-15 min)
    Stop the timer and leave the animal without disturbing it for 10 min (Figure 2).
  10. Phase II (15-30 min)
    Set the timer for 15 min and start it to mark the beginning of the phase II (Figure 2). Use a chronometer to visually measure the total time the animal spends licking or biting the injected paw during this period (15 min) (Video 1). Annotate the result.
  11. Finishing
    Return the animal to its home cage.
    Note: If a recording system is used switch off the camera. Check whether the animal needs any special attention or health care (i.e., bleeding, extra tissue damage, etc.).
  12. Cleaning
    Clean all the material used.
    Note: Repeat the experiment with another animal if required. If a camera is used, visualize the videos and measure the total time the animal spends licking or biting the injected paw during each period (phase I and II) (Video 1).

    Video 1. Video showing formalin-induced hind paw licking in mouse

Data analysis

The antinociception induced by drug treatment in the formalin test can be calculated by the following formula:

where, LTV and LTD represent the licking/biting time in the vehicle- and drug-treated animals, respectively.
The antinociceptive effect is expressed as a percentage (mean ± SEM) of the maximum effect observed (Figure 4). The results are analysed either by Student’s t-test or by one-way analysis of variance (ANOVA) followed by Dunnett’s post-hoc test using vehicle-treated animals as a control (Figure 4). Statistical significance was set as P < 0.05.

Figure 4. Representative results. Antinociceptive effect of raseglurant (10 mg/kg) as analgesic drug using the formalin test. ***P < 0.001 Student’s t-test when compared to the vehicle (physiological saline solution) treated animals (see Figure 2). Extracted from Font et al., 2017.


  1. 2.5 %formalin solution (corresponds to a 0.93 % formaldehyde in physiological saline solution)
    25 µl formaldehyde solution (36.5-38% in H2O)
    975 µl sterile physiological saline solution


This work was supported by MINECO/ISCIII (SAF2014-55700-P and PIE14/00034), the Catalan government (2014 SGR 1054), ICREA (ICREA Academia-2010), Fundació la Marató de TV3 (Grant 20152031) and IWT (SBO-140028) to FC. MINECO (PCIN-2013-017-C03-01 and CTQ2014-57020-R), the Catalan government (2014SGR109 and 2014CTP0002) to AL. ERANET Neuron project ‘LIGHTPAIN’. This protocol was adapted from previous work: Font et al., 2017. The authors declare not conflict of interest.


  1. 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.
  2. 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.
  3. 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.
  4. Mogil, J. S. (2009). Animal models of pain: progress and challenges. Nat Rev Neurosci 10(4): 283-294.
  5. Tjolsen, A., Berge, O. G., Hunskaar, S., Rosland, J. H. and Hole, K. (1992). The formalin test: an evaluation of the method. Pain 51(1): 5-17.






关键字:疼痛, 伤害感受, 炎症, 镇痛, 小鼠


  1. 注射器(BD Micro-Fine TM黛咪,U-100胰岛素,30G×1/2“-0.33×8mm)(BD,目录号:324826)(图1d)
  2. Eppendorf管(图1b)
  3. 动物:使用8周龄,体重20-25g的成年雄性CD-1小鼠(Charles River Laboratories,L'Arbresle,France; RRID:MGI:3785721)
    注意:将动物饲养在标准笼子中,可自由获取食物和水,并将其维持在受控的标准条件下(从7:30 AM,22°C和66%湿度开始的12小时黑暗/光照循环) 。所有操作在9:00至16:00之间进行。本研究中的程序是根据国家卫生研究院的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等人。,1997)和欧盟指令(2010/63 / EU)。有关机构的伦理委员会(CEUA / UFSC和CEEA / UB)批准了该议定书。努力减少痛苦,减少实验中使用的动物的数量。为达到统计学显着性,强烈建议每组至少10只小鼠。
  4. 70%乙醇
  5. 福尔马林(H 2 O中36.5-38%甲醛溶液)(Sigma-Aldrich,目录号:F8775-500ML) 注:用于制备生理盐水溶液中的最终2.5%福尔马林
  6. 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)。
  7. 车辆。无菌生理盐水(0.9%NaCl)溶液(Grifols,目录号:605137)
  8. 无菌清洁解决方案
    注意:使用H 2 O中的70%乙醇。
  9. 2.5%福尔马林溶液(见食谱)


  1. 观察室
    不得进入食物或饮水 注意:可以选择使用录制系统(即摄像机)。因此,将记录系统放置在观测室的后面,下面和/或上方。确保动物的后爪在所有观察角度都清晰可见,因此观察室周围的一组镜子可用于此目的。
  2. 称重机(图1c)
  3. 彩色表面(图1e)
  4. 抑制小鼠的装置(图1f)
  5. PIPETMAN经典TM吸管(Gilson,型号:P20,P200和P1000,分别为F123600,F123601和F123602)
  6. 定时器(图1g)和精密时计(图1h)
  7. 个人防护装备(图1i)(实验室外套,手套,口罩,等)

    图1.福尔马林测试所需的材料。 (b),余量(c),胰岛素注射器(d),黑色塑料表面(e),小鼠抑制器(f),计时器(g) ,天文钟(h)和手套(i)。


  1. 习惯
  2. 试剂准备
  3. 称量动物
  4. 镇痛药的施用
    (腹腔注射,腹腔内注射)待测的镇痛药(即em / g,raseglurant;图2)或相同体积的载体(生理盐水溶液),使用胰岛素注射器(图1d)。
    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。
  5. 适应


  6. 福尔马林加载
  7. 福尔马林管理
    1. 将针头放在脚趾和脚踝之间,插入皮肤表面之下,避免任何组织损伤。
    2. 注射应该坚定和时间优化。动物可以将这种抑制看作是压力或刺激,并且可以增加结果的可变性。


  8. 第一阶段(0-5分钟)
    1. 如果使用录音系统,请相应地打开和关闭系统。
    2. 我们强烈建议研究者在这一步进行一些训练,以熟悉舔/咬的行为。
  9. 静止期(5-15分钟)
  10. 第二阶段(15-30分钟)
  11. 整理
  12. 清洁




抗伤害感受作用表示为观察到的最大效应的百分比(平均值±SEM)(图4)。通过Student's t检验或通过单因素方差分析(ANOVA),随后使用载体处理的动物作为对照的Dunnett事后检验(图4)来分析结果。统计学显着性被设定为 P 0.05。

图4.有代表性的结果使用福尔马林试验,作为止痛药的抗恶性肿瘤作用(10mg / kg)。 *** P &lt; 0.001与运载体(生理盐水溶液)处理的动物相比,学生的测试(见图2)。摘自Font et al。,2017。


  1. 2.5%福尔马林溶液(对应于生理盐水中0.93%的甲醛)
    25μl甲醛溶液(36.5-38%于H 2 O中)


这项工作得到了MINECO / ISCIII(SAF2014-55700-P和PIE14 / 00034),加泰罗尼亚政府(2014 SGR 1054),ICREA(ICREA Academia-2010),FundaciólaMaratóTV3(Grant 20152031)和IWT(SBO -140028)到FC。 MINECO(PCIN-2013-017-C03-01和CTQ2014-57020-R),加泰罗尼亚州政府(2014SGR109和2014CTP0002)至AL。 ERANET神经元项目“LIGHTPAIN”。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. 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。
  2. Dubuisson,D。和Dennis,S.G。(1977)。 福尔马林试验:定量研究吗啡,哌替啶和脑干刺激的镇痛作用大鼠和猫。 4(2):161-174。
  3. 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.
  4. Mogil,J.S。(2009)。 痛苦的动物模型:进步和挑战 Nat Rev Neurosci 10(4):283-294。
  5. Tjolsen,A.,Berge,O. G.,Hunskaar,S.,Rosland,J.H。和Hole,K。(1992)。 福尔马林试验:评估方法。 > 51(1):5-17。
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Copyright López-Cano et al. This article is distributed under the terms of the Creative Commons Attribution License (CC BY 4.0).
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. López-Cano, M., Fernández-Dueñas, V., Llebaria, A. and Ciruela, F. (2017). Formalin Murine Model of Pain. Bio-protocol 7(23): e2628. DOI: 10.21769/BioProtoc.2628.
  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.