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Assessment of Thermal Pain Sensation in Rats and Mice Using the Hargreaves Test
使用Hargreaves测试法评估大鼠和小鼠的热疼痛感   

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参见作者原研究论文

本实验方案简略版
The Journal of Neuroscience
Jul 2016

Abstract

The Hargreaves test is specifically designed to assess thermal pain sensation in rodents such as rats and mice. This test has been used in experiments involving pain sensitization or recovery of thermal pain response following neural injury and regeneration. We present here a step-by-step protocol highlighted with important notes to guide first-time users through the learning process. Additionally, we have also included representative data from a rat model of sensory denervation showing how the data can be analysed to obtain meaningful results. We hope that this protocol can also assist potential users in deciding whether the Hargreaves test is a suitable test for their experiment.

Keywords: Thermal pain (热疼痛), Hargreaves (Hargreaves), Rats (大鼠), Mice (小鼠), Behavioral testing (行为测试)

Background

The somatosensory system is responsible for processing sensory stimuli received from the environment. These sensory stimuli include pain, touch, pressure and vibration. To study how these stimuli are processed with the ultimate goal of repairing the system in the event of injury, neuroscientists have used a plethora of animal models and behavioral tests. Some of these experiments include administration of a noxious substance into the nervous system to investigate increased pain sensitivity (Zurowski et al., 2012), inducing a central or peripheral injury to the nervous system and then observing whether particular treatments promote neural regeneration (Andrews et al., 2009; Tan et al., 2012; Cheah et al., 2016), or developing in vivo neurodegenerative models with pathology in sensory neurons (Mellone et al., 2013). Whichever the case, having a suitable test to study the behavioral response of the animals is key.

Of all the sensations, pain is perhaps one of the most studied. Transmitted via different nociceptors, pain can be further categorized into different modalities such as thermal, mechanical and chemical. Depending on the experimental goal, all of these modalities can be assessed with a specific behavioral test. We present here our approach to the Hargreaves test which is a behavioral test designed for assessing response to thermal pain in rodents such as rats and mice (Hargreaves et al., 1988). Based on our experience, the Hargreaves test is relatively straight-forward and novel users can master it in a short period of time. With the aid of this step-by-step protocol, we hope to assist potential users in deciding if the Hargreaves test is a suitable test for them and also guide them through the learning process in an efficient manner.

Materials and Reagents

  1. Tissue paper
    Note: The animals may urinate or defecate in the enclosure. Any fluid found on the framed glass panel should be cleaned immediately as this can affect the thermal conductance of the glass panel.
  2. Disinfectant cleaning solution (e.g., 70% ethanol)
    Use a disinfectant cleaning solution to remove animal odor, urine and/or feces after each session
  3. Animals
    Note: The activity level of each animal strain can affect the test tremendously as proper plantar placement of the paws is required for the test to be performed with reliable results. For whichever strain is chosen, habituation to the enclosure should be performed in the week prior to the start of experiment in order to acclimatize the animals to the setup as well as to take baseline readings.
    1. For rats, the more docile Lewis strain may be ideal for the test because they have a tendency to move about their enclosures minimally compared to other strains, however they may fall asleep during the test resulting in delayed responsiveness. On the other hand, the more active Lister-Hooded strain may move too much, hence making the test difficult and/or potentially invalidating the results. Sprague Dawley rats have a tendency to have moderate activity level and are thus another ideal strain for this test.
    2. For mice, the C57BL/6 strain is generally more active than other strains such as BALB/c. The more active strains may make progressing with the test difficult although with sufficient acclimatization to the testing apparatus, any strain can be used.
  4. Sucrose/food pellets
    Optional: Feeding animal a small handful (~5-10) of sucrose/food pellets may be helpful in reducing the movement and calming the animals in their enclosure.

Equipment

  1. Hargreaves test (Ugo Basile, catalog number: 37370 or Harvard Apparatus, catalog number: 72-6692 )
    A complete set of the Hargreaves test should comprise the following components (Figures 1-3):
    1. Controller
      For manipulation of test settings such as infrared intensity, display of reaction time, and power input port
    2. Emitter/detector vessel
      With a fibre optic cable connecting to the controller for infrared emission and paw movement detection
    3. Framed glass panel
      For optimal thermal conductance, the glass panel should be kept clean and protected from damage
    4. Animal enclosure
      Large: holds up to 6 rats at a time
      Small: holds up to 12 mice at a time
    5. Large platform
    6. Supporting columns


      Figure 1. A complete setup of the Hargreaves test


      Figure 2. The Hargreaves test controller and emitter/detector vessel


      Figure 3. Animal enclosure. The large enclosure can hold up to 6 rats, one animal in each compartment, while the small enclosure can hold up to 12 mice.

    Procedure

    Note: All of the following animal procedures were conducted in accordance with the United Kingdom Animals (Scientific Procedures) Act 1986. Please check with local/national regulations before starting the protocol.

    1. Choose a suitable room for behavioral assessment.
      Note: External stimuli such as noise and vibration in the environment can cause anxiety in the animals. They may exhibit excessive movements if they are anxious or freeze if they are frightened. In either case, this would make progressing with the test difficult. It is recommended to choose a calm and quiet room for the test, and preferably somewhere without any disruptions during the test.
    2. Set up the equipment as shown in Figure 1.
    3. Habituate the animals to the testing chamber.
      1. Transfer the animals from the holding room to the behavioral room in a calm manner.
      2. Gently place each animal into its own compartment of the enclosure.
      3. Acclimatize the animals to the enclosure for 30-60 min.
        Note: The animals may show curiosity to the new environment by moving around in the enclosure for the first 10-15 min, after which they should calm down. Placing sucrose/food pellets in the enclosure may be helpful in calming them down.
      4. Return the animals back to the holding room and repeat the habituation step for at least 2 days before performing the actual Hargreaves test.
      5. Thoroughly clean the enclosure and framed glass panel after each use.
    4. Perform the Hargreaves test.
      It is recommended that a baseline reading be obtained before starting the experiment. This step also allows the experimenter to optimize the test for each particular experiment and animal strain.
      1. Transfer the animals from the holding room to the enclosure as before.
      2. Acclimatize the animals in the enclosure for 15-20 min or until they are calm.
        Note: The animals may fall asleep while being acclimatized or during the test. Gently tap the enclosure to wake them up before performing the test if necessary.
      3. Set the desired infrared intensity (Figure 4).
        Note: This may require optimization before starting the experiment. 48 units was used for our experiment with adult male Lewis rats.


        Figure 4. Controller. The controller contains a display for reaction time in seconds, the START button and adjustable infrared intensity.

      4. Position the infrared emitter/detector on the vessel to directly underneath the center of the paw being tested (Figures 5-6).
        Note: The guiding lines on the vessel should be very useful for this step and there must be proper plantar placement of the paw for the test to be valid.


        Figure 5. Emitter/detector vessel. The guiding lines on the vessel are useful in positioning the infrared emitter/detector directly underneath the center of the paw.


        Figure 6. The correct positioning of the paw. The infrared emitter/detector should be positioned directly underneath the center of the paw (white arrows). There must be proper plantar placement of the paw for the test to be valid.

      5. Start the test by pressing the START button.
        Note: The timer should start automatically at the same time as the infrared light is being switched on.
      6. Observe the animal’s paw until the animal elicits a withdrawal response due to sensing of thermal heat.
        Note: Movement of the paw will be detected by the vessel, and the controller will automatically switch off the infrared light and stop the timer altogether. If the animal moves the paw voluntarily (not as a reaction to the heat), this is not a withdrawal response and the particular trial should not be included. Typical withdrawal response due to the heat should be accompanied by the animal checking/observing or licking their paw. (Video 1)

        Video 1. Typical withdrawal response in rats. This video clip shows two different withdrawal scenarios of a rat that can occur whilst the animal is in the testing apparatus. The first example shows a proper withdrawal response in which the animal removes its paw from the heat and immediately licks its paw as a reflex. The second example shows removal of the paw from the heat source which is not due to the heat but rather is voluntary movement of the paw. In the second example there is no licking of the paw or acknowledgement of the heat and should therefore not be included as a proper withdrawal response.

      7. Record the reaction time.
        Note: Depending on local/national regulations, it is recommended that the trial should be terminated if the animal fails to respond within 20 sec to avoid potential burn injury.
      8. Repeat the test for at least 3 trials* to obtain an average reaction time.
        Notes:
        1. *If time permits, 5 trials are recommended. In this case, the lowest and highest values can then be discounted to remove outlying values.
        2. Allow for approximately 5 min of interval time before repeating the test on the same animal. The experimenter can test other animals in the enclosure during this interval.
      9. Carefully return the animals back to the holding room.
      10. Thoroughly clean the enclosure and framed glass panel after each use.
      11. Repeat steps 4a-4j at the next experimental data point if required.

    Data analysis

    The amount of time required to elicit a withdrawal response is termed as withdrawal latency which is measured in seconds. A longer withdrawal latency signifies a slower withdrawal response and vice versa. To obtain the average reaction time for an animal, calculate the mean from at least 3 trials. If 5 trials have been performed, the lowest and the highest values can be discounted as outlying values. To obtain a data point for a group of animals with one animal represents n = 1, calculate the mean of the average reaction time of the animals in the group. If the same set of animals is being tested repeatedly over different experimental conditions or timepoints, a paired or repeated measures statistical test should be used to determine the statistical significance. Usually, a P value of less than 0.05 is deemed to be statistically significant. Representative data are shown below (Figure 7).


    Figure 7. Representative Hargreaves test data for adult Lewis rats which underwent a quadruple (C5-C8) dorsal root crush injury of the left forepaw or sham surgery. A baseline reading was recorded a week before surgery and experimental readings were recorded weekly during 1-8 weeks after surgery. As opposed to the sham control group, a longer time (in seconds) was required to elicit a withdrawal response from the injured group a week after surgery and this was sustained throughout the duration of the experiment signifying chronic thermal pain sensory deficit. The data were analyzed by repeated measures ANOVA and expressed as mean ± SEM, *P < 0.05 was statistically significant, n = 4.

    Notes

    1. It is important that the experimenter watch the animals closely during the test to make sure they do not remove their paw because they are moving it voluntarily, as opposed to being a reaction to the heat. If this occurs, the particular trial should be voided as the withdrawal response observed was likely not in response to the heat. The Hargreaves device itself cannot make the distinction whether the movement is due to the heat or is voluntary. Typical withdrawal response due to the heat should be accompanied with the animals checking or licking their paw.
    2. In terms of animal welfare, please handle the animals very gently. Animals subject to the Hargreaves test may have hypersensitivity to thermal pain due to a procedure such as injection of a hyperalgesic substance as part of the experiment, or reduced pain sensation due to receiving a peripheral or central nervous system injury previously. In the event of reduced pain sensation, a cut-off point (recommended: 20 sec) should be set for the test so the animals do not suffer from potential burn injury.

    Acknowledgments

    M.R.A. is supported by a research grant from the Biotechnology and Biological Sciences Research Council (BBSRC). J.W.F. is supported by a grant from the European Union/Czech Ministry of Education–Operation Programme Research (CZ.02.1.01/0.0/0.0/15_003/0000419).

    References

    1. Andrews, M. R., Czvitkovich, S., Dassie, E., Vogelaar, C. F., Faissner, A., Blits, B., Gage, F. H., ffrench-Constant, C. and Fawcett, J. W. (2009). α9 integrin promotes neurite outgrowth on tenascin-C and enhances sensory axon regeneration. J Neurosci 29(17): 5546-5557.
    2. Cheah, M., Andrews, M. R., Chew, D. J., Moloney, E. B., Verhaagen, J., Fassler, R. and Fawcett, J. W. (2016). Expression of an activated integrin promotes long-distance sensory axon regeneration in the spinal cord. J Neurosci 36(27): 7283-7297.
    3. Hargreaves, K. M., Dubner, R., Brown, F., Flores, C. and Joris, J. (1988). A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain 32: 77-88.
    4. Mellone, M., Kestoras, D., Andrews, M.R., Dassie, E., Crowther, R.A., Stokin, G.B., Tinsley, J., Horne, G., Goedert, M., Tolkovsky, A. M. and Spillantini, M. G. (2013). Tau pathology is present in vivo and develops in vitro in sensory neurons from human P301S tau transgenic mice: a system for screening drugs against tauopathies. J Neurosci 33(46): 18175-18189.
    5. Tan, C. L., Andrews, M. R., Kwok, J. C., Heintz, T. G., Gumy, L. F., Fassler, R. and Fawcett, J. W. (2012). Kindlin-1 enhances axon growth on inhibitory chondroitin sulfate proteoglycans and promotes sensory axon regeneration. J Neurosci 32(21): 7325-7335.
    6. Zurowski, D., Nowak, L., Machowska, A., Wordliczek, J. and Thor, P. J. (2012). Exogenous melatonin abolishes mechanical allodynia but not thermal hyperalgesia in neuropathic pain. The role of the opioid system and benzodiazepine-gabaergic mechanism. J Physiol Pharmacol 63(6): 641-647.

简介

Hargreaves测试专门用于评估鼠和老鼠等啮齿动物的热痛感。 该试验已被用于涉及疼痛敏化或神经损伤和再生后热痛反应恢复的实验。 我们在这里介绍一个循序渐进的协议,重点介绍了首次使用者通过学习过程。 此外,我们还包括来自感官去神经支配的大鼠模型的代表性数据,显示如何分析数据以获得有意义的结果。 我们希望该协议还可以帮助潜在用户决定Hargreaves测试是否是他们实验的合适测试。
【背景】体感系统负责处理从环境中获得的感觉刺激。这些感觉刺激包括疼痛,触觉,压力和振动。为了研究如何处理这些刺激物,最终目的是在受伤的情况下修复系统,神经科学家已经使用了大量的动物模型和行为测试。这些实验中的一些包括向神经系统施用有害物质以调查增加的疼痛敏感性(Zurowski等人,2012),诱导神经系统的中枢或外周损伤,然后观察特定治疗是否促进神经再生(Andrews et等等,2009; Tan et al。,2012; Cheah et al。,2016),或发展了具有感觉神经元病理学的体内神经变性模型(Mellone等,2013)。无论哪种情况,都有适当的测试来研究动物的行为反应是关键。
   在所有的感觉中,疼痛可能是最受研究的之一。通过不同的伤害感受器传播,疼痛可以进一步分为不同的形式,如热,机械和化学。根据实验目标,所有这些模式都可以通过特定的行为测试进行评估。我们在这里介绍我们对Hargreaves测试的方法,这是一种行为测试,旨在评估鼠类(如老鼠和小鼠)对热痛苦的反应(Hargreaves等,1988)。根据我们的经验,Hargreaves测试相对简单,新颖的用户可以在短时间内掌握它。借助这个一步一步的协议,我们希望协助潜在用户决定哈格里夫斯考试是否适合他们,并以有效的方式引导他们学习过程。

关键字:热疼痛, Hargreaves, 大鼠, 小鼠, 行为测试

材料和试剂

  1. 组织纸
    注意:动物可能会在围栏内排尿或排便。框架玻璃面板上发现的任何液体都应立即进行清洁,因为这可能会影响玻璃面板的导热性。
  2. 消毒剂清洁溶液(例如,70%乙醇)
    每次使用消毒液清洗液,以清除动物的气味,尿液和/或粪便
  3. 动物
    注意:每个动物菌株的活动水平可以极大地影响测试,因为需要执行适当的足底放置爪以进行可靠的测试。对于选择哪种菌株,应在实验开始前的一周内对外壳进行习性,以使动物适应该设置以及进行基线读数。
    1. 对于老鼠来说,更温和的Lewis菌株对于测试来说可能是理想的,因为它们与其他菌株相比具有最小化的趋势,但是在测试期间它们可能会入睡,导致延迟反应。另一方面,更有活力的Lister-Hooded菌株可能移动太多,因此使测试难以和/或潜在地使结果无效。 Sprague Dawley大鼠具有中度活动水平的倾向,因此是该测试的另一个理想的应变
    2. 对于小鼠,C57BL / 6株通常比其他菌株如BALB / c更具活性。更有活力的菌株可能会随着测试难以进展,尽管足够适应测试设备,但可以使用任何菌株。
  4. 蔗糖/食品颗粒
    可选:将少量(〜5-10)的蔗糖/食物颗粒饲喂动物可能有助于减少其外壳中的动物的运动和平静。

设备

  1. Hargreaves测试(Ugo Basile,目录号:37370或Harvard Apparatus,目录号:72-6692)
    一整套Hargreaves测试应包括以下组件(图1-3):
    1. 控制器
      用于操作诸如红外线强度,反应时间显示和电源输入端口等测试设置
    2. 发射器/检测器容器
      使用连接到控制器的光纤电缆进行红外发射和爪子运动检测
    3. 框架玻璃面板
      为了获得最佳的导热性,玻璃面板应保持清洁,防止损坏
    4. 动物围栏
      大:一次最多可容纳6只老鼠 小:一次最多可容纳12只小鼠
    5. 大平台
    6. 支持栏


      图1. Hargreaves测试的完整设置


      图2. Hargreaves测试控制器和发射器/探测器容器


      图3.动物围栏大型围栏最多可容纳6只大鼠,每个隔间有一只动物,而小外壳可容纳12只小鼠。

    程序

    注意:以下所有动物手术均按照1986年英国动物(科学程序)法进行。请在开始协议之前查阅本地/国家规定。

    1. 选择一个适合行为评估的房间。
      注意:外部刺激如环境中的噪音和振动会引起动物的焦虑。如果他们害怕,他们可能会表现出过度的运动,如果他们受到惊吓,就会冻结。在任何一种情况下,这将使测试困难进行。建议选择一个平静安静的房间进行测试,最好在测试过程中没有任何中断的地方。
    2. 设置如图1所示的设备。
    3. 将动物习惯于测试室。




      1. 轻轻地将每只动物放在自己的封闭空间中。
      2. 将动物适应环境30-60分钟。
        注意:这些动物可能会通过在前10-15分钟的围墙内移动,向新的环境展现好奇心,之后他们应该冷静下来。将蔗糖/食物颗粒放入外壳可能有助于使它们平静下来。
      3. 在执行实际的Hargreaves测试之前,将动物返回到保持室并重复习惯步骤至少2天。
      4. 每次使用后,彻底清洁外壳和框架玻璃面板。
    4. 执行Hargreaves测试。
      建议在开始实验之前获得基线读数。该步骤还允许实验者优化每个特定实验和动物菌株的测试。
      1. 将动物从前往搬运室转移到围栏。
      2. 适应环境中的动物15-20分钟,或直到平静 注意:在适应环境或试验期间,动物可能会睡着。轻轻敲击机箱,以便在执行测试之前将其唤醒,如有必要。
      3. 设置所需的红外线强度(图4) 注意:这可能需要在开始实验之前进行优化。我们用成年雄性Lewis大鼠进行48个单位的实验。


        图4.控制器。 控制器包含反应时间(以秒为单位)的显示,START按钮和可调节的红外线强度。

      4. 将红外线发射器/探测器放置在容器上直接位于被测爪的中心的下方(图5-6)。
        注意:船上的引导线对于该步骤应该非常有用,并且必须有适当的足底放置爪子才能使测试有效。


        图5.发射器/检测器容器。 船上的引导线可用于将红外发射器/探测器直接放置在爪中心的下方。


        图6.爪子的正确定位。 红外线发射器/检测器应直接定位在爪子中心的正下方(白色箭头)。必须有足够的足底放置爪子才能使测试有效。

      5. 按开始按钮开始测试。
        注意:定时器应在红外线开启的同时自动启动。
      6. 观察动物的爪子,直到动物由于感觉到热量而引起撤离反应。
        注意:由船只检测到爪子的移动,控制器将自动关闭红外线并完全停止计时器。如果动物自愿移动爪子(而不是对热量的反应),这不是撤离反应,不应包括特定的试验。由于热量引起的典型的撤离反应应伴有动物检查/观察或舔其爪子。 (视频1)

        Video 1. Typical withdrawal response in rats. This video clip shows two different withdrawal scenarios of a rat that can occur whilst the animal is in the testing apparatus. The first example shows a proper withdrawal response in which the animal removes its paw from the heat and immediately licks its paw as a reflex. The second example shows removal of the paw from the heat source which is not due to the heat but rather is voluntary movement of the paw. In the second example there is no licking of the paw or acknowledgement of the heat and should therefore not be included as a proper withdrawal response.

        To play the video, you need to install a newer version of Adobe Flash Player.

        Get Adobe Flash Player


      7. 记录反应时间。
        注意:根据当地/国家规定,建议如果动物在20秒内未能作出反应以避免潜在的烧伤,建议终止试验。
      8. 重复测试至少3次试验*以获得平均反应时间。
        注意:
        1. 如果时间允许,建议进行5次试验。在这种情况下,最低和最高值可以被打折以消除偏离的价值。
        2. 在同一动物重复测试前,允许间隔时间约5分钟。实验者可以在此间隔期间对外壳中的其他动物进行测试。
      9. 仔细地将动物带回保管室。
      10. 每次使用后,彻底清洁外壳和框架玻璃面板。
      11. 如果需要,在下一个实验数据点重复步骤4a-4j。

    数据分析

    引出撤回响应所需的时间量称为撤回延迟,以秒为单位。更长的提款延迟表示较慢的提款响应,反之亦然。为了获得动物的平均反应时间,从至少3次试验中计算平均值。如果进行了5次试验,则最低和最高值可以作为离岸价值打折扣。为了获得具有一只动物的一组动物的数据点代表n = 1,计算组中动物的平均反应时间的平均值。如果在不同的实验条件或时间点上重复测试同一组动物,则应使用配对或重复测量统计检验来确定统计学显着性。通常,低于0.05的 P 值被认为具有统计学意义。代表性资料如下(图7)

    图7.代表性的Hargreaves测试数据,用于成年Lewis大鼠,其经历左前爪或假手术的四重(C5-C8)背根压伤。在术前和实验一周记录基线读数手术后1-8周每周记录一次。与假手术组相反,手术后一周需要较长时间(以秒为单位)从受伤组引出戒断反应,并且在实验持续期间持续表明慢性热痛感觉缺陷。通过重复测量方差分析来分析数据,表示为平均值±SEM,* P < 0.05有统计学意义,n = 4。

    笔记

    1. 重要的是,实验者在测试期间密切观察动物,以确保它们不会自动移动它们,因为它们是自动移动的,而不是对热的反应。如果发生这种情况,特定的试验应该被废除,因为观察到的撤回反应可能不会响应热量。哈格里夫装置本身不能区分运动是由于热还是自愿的。典型的由于热量引起的戒断反应应伴随动物检查或舔其爪子。
    2. 在动物福利方面,请妥善处理动物。受Hargreaves试验影响的动物可能由于诸如注射高痛药物作为实验的一部分而引起的热痛感过敏,或者由于以前受到外周或中枢神经系统损伤而引起的疼痛感减轻。在疼痛感减轻的情况下,应设置一个截止点(推荐:20秒)以进行测试,以免动物遭受潜在的烧伤。

    致谢

    M.R.A.得到了生物技术与生物科学研究理事会(BBSRC)的研究资助。 J.W.F.得到欧盟/捷克教育部 - 行动计划研究部(CZ.02.1.01 / 0.0 / 0.0 / 15_003 / 0000419)的资助。

    参考

    1. Andrews,MR,Czvitkovich,S.,Dassie,E.,Vogelaar,CF,Faissner,A.,Blits,B.,Gage,FH,ffrench-Constant,C.and Fawcett,JW(2009)α9整联蛋白促进腱生蛋白C上的神经突生长,并增强感觉轴突再生。 J Neurosci 29(17):5546-5557。
    2. Cheah,M.,Andrews,MR,Chew,DJ,Moloney,EB,Verhaagen,J.,Fassler,R.and Fawcett,JW(2016)。 激活的整联蛋白的表达促进脊髓中的长距离感觉轴突再生。 Neurosci 36(27):7283-7297。
    3. Hargreaves,KM,Dubner,R.,Brown,F.,Flores,C.and Joris,J。(1988)。 一种用于测量皮肤痛觉过敏热敏感伤害的新方法。 32:77-88。
    4. Mellone,M.,Kestoras,D.,Andrews,MR,Dassie,E.,Crowther,RA,Stokin,GB,Tinsley,J.,Horne,G.,Goedert,M.,Tolkovsky,AM and Spillantini,MG( 2013)。 Tau病理存在体内,并且在来自人P301S tau转基因小鼠的感觉神经元中的体外发展:用于筛选针对tau蛋白病的药物的系统。 Neurosci 33 (46):18175-18189。
    5. Tan,CL,Andrews,MR,Kwok,JC,Heintz,TG,Gumy,LF,Fassler,R。和Fawcett,JW(2012)。 Kindlin-1可增强硫酸软骨素蛋白聚糖上的轴突生长,并促进感觉轴突再生。 Neurosci 32(21):7325-7335。
    6. Zurowski,D.,Nowak,L.,Machowska,A.,Wordliczek,J.and Thor,PJ(2012)。外源性褪黑素消除机械性异常性疼痛,但不是神经性疼痛中的热痛觉过敏。阿片类药物系统和苯二氮卓类药物机制的作用。生理药理学63(6):641-647。
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Copyright: © 2017 The Authors; exclusive licensee Bio-protocol LLC.
引用: Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
  1. Cheah, M., Fawcett, J. W. and Andrews, M. R. (2017). Assessment of Thermal Pain Sensation in Rats and Mice Using the Hargreaves Test. Bio-protocol 7(16): e2506. DOI: 10.21769/BioProtoc.2506.
  2. Cheah, M., Andrews, M. R., Chew, D. J., Moloney, E. B., Verhaagen, J., Fassler, R. and Fawcett, J. W. (2016). Expression of an activated integrin promotes long-distance sensory axon regeneration in the spinal cord. J Neurosci 36(27): 7283-7297.
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