This continues until at least four readings are obtained after the first change of direction, and the sequence of outcomes ( for no response or + for response) is recorded (Figure 2A). In human patients, a distinction is made between stimulus-evoked pain and stimulus-independent or spontaneous pain. Address: 2200 Smithtown Avenue This sample - and the results - are biased, as most workers are at their jobs during these hours. His experiments, conducted in what is known as "Skinner's box," are some of the most well-known experiments in psychology. (1980). This raises a moral issue in animal experimentation. Twitter (C) The percent response method. Joint Bone Spine 72, 359371. The Rat Grimace Scale is able to detect spontaneous pain induced by intraarticular kaolin-carrageenan, intraplantar Complete Freunds Adjuvant (CFA), and post-surgical pain. The cold plate test is one of the simplest assays to determine behavioral responses to both noxious and innocuous cold temperatures in both mice and rats. The amount of substrate displaced over a set period of time is recorded. ILAR J. 89, 707758. A survey posted only on its website limits the number of people who will participate to those who already had an interest in their products, which causes a voluntary response bias. Despite these advantages of automation, experimenters still need to be experienced to distinguish true responses from touch-on responses and ambulation. In unilateral pain models, many changes in gait parameters are observed, including reduced intensity (paw pressure), reduced print area, reduced stance phase duration (time spent on paw), increased swing phase duration (time spent off paw) in the ipsilateral hind paw compared to contralateral hind paw, consistent with reduced weight bearing and guarding behaviors (Parvathy and Masocha, 2013; Yin et al., 2016). Randall-Selitto test: a new approach for the detection of neuropathic pain after spinal cord injury. doi: 10.1002/art.21403, Miller, A. L., and Leach, M. C. (2015). Pain 16, 485495. (C) Randall-Selitto test (handheld device). 111, 409419. doi: 10.1038/nprot.2006.19, Decosterd, I., and Woolf, C. J. Weight distribution between the hind paws is recorded. However the distinction between stimulus-evoked and non-stimulus evoked pain may be difficult to make clinically, as arguable it could be allodynia occurring from an unidentified stimulus. These decades of work will enable the integration of new techniques and technologies for cerebral palsy research and maximize the utility of the hypoxia-ischemia model. LinkedIn. Efficient analysis of experimental observations. By comparing the frequencies of behaviors in animal models of pain as opposed to control states, inference about different pain states (and especially spontaneous nociception) can be made. This book condenses information from many sources on topics related to the care and use of rats in research. The Wistar outbred albino rat is perhaps the most popular rat model due to its multi-purpose characteristics, making it a powerful species for medical research into infectious diseases, or as a surgical model. 9, 8998. In fact, over 90% of test subjects are mice, birds and rats. Similarly, as it cannot be said that the animal feels pain, analgesia and analgesic intervention cannot take placeonly anti-nociception and anti-nociceptive interventions can. Mice are placed individually in small cages with a barred floor. TRPM3 is a nociceptor channel involved in the detection of noxious heat. Accordingly, as pain cannot be directly measured in rodents, it has been necessary to develop indirect methods to quantify and evaluate pain-like behaviors in non-anesthetized animals which are reliable, reproducible, sensitive and specific (Mogil, 2009). Neurosci. Use of dynamic weight bearing as a novel end-point for the assessment of Freunds Complete Adjuvant induced hypersensitivity in mice. Pain Headache Rep. 18:456. doi: 10.1007/s11916-014-0456-x, Jensen, T. S., and Finnerup, N. B. Figure 1. The use of ultrasonic (inaudible) vocalization as an endpoint has also been studied, however it has not consistently been shown to increase in response to noxious stimuli (Han et al., 2005; Wallace et al., 2005; Williams et al., 2008). Neuron 54, 379386. Ronkonkoma, NY 11779, 2023 ingenious targeting laboratory. Irwin, S., Houde, R. W., Bennett, D. R., Hendershot, L. C., and Seevers, M. H. (1951). Characterization of the foot withdrawal response to noxious radiant heat in the rat. doi: 10.1213/ane.0000000000000291, Carlton, S. M., Lekan, H. A., Kim, S. H., and Chung, J. M. (1994). This assay is based on the hypothesis that a rodent with spontaneous pain will guard the painful paw, leading to changes in its gait (exhibiting a limp or changes in stride size, for instance) in addition to changes in weight bearing (Jacobs et al., 2014). While application of a contact heat stimulus achieves consistent and efficient thermal transfer, it also represents a mechanical stimulus that may lead to premature paw withdrawal in models with mechanical allodynia. Methods 113, 9197. (2000). Pain 32, 7788. Lancet Neurol. 67, 705713. A radiant or infrared heat source is positioned underneath the animal and aimed at the plantar surface of the hind paw (Figure 3C). Behav. The Randall-Selitto or paw pressure test was developed as a tool to assess response thresholds to mechanical pressure stimulation and is often considered a measure of mechanical hyperalgesia (Figure 1C; Randall and Selitto, 1957). It should be noted that rather than flinching or licking, some rat strains tend to simply avoid weight bearing on the affected paw or reposition their stance to minimize contact with the cool surface, so all observation should be adapted to the specific model animal. Biol. J. Pharmacol. A cold stimulus is applied to the hind paw using a cut off syringe filled with dry ice or wet ice and the time to paw withdrawal is recorded. doi: 10.1213/01.ane.0000158471.41575.f0, Vriens, J., Owsianik, G., Hofmann, T., Philipp, S. E., Stab, J., Chen, X., et al. Undoubtedly, nociception, or the ability to detect a potentially harmful stimulus, is a fundamental physiological function in mammals and indeed many other species. The tail flick test, first described in 1941, involves application of a heat stimulus to the tail of mice and rats, and the time taken for the tail to flick or twitch is recorded (DAmour and Smith, 1941; Figure 3A). J. Neurosci. The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. It also demonstrates two additional concepts, originally outlined by Pavlov. Clin. Circadian pattern of spontaneous behavior in monarthritic rats: a novel global approach to evaluation of chronic pain and treatment effectiveness. Stimulus-independent or spontaneous pain may be paroxysmal (sudden and severe) or continuous, and can be described as aching, cramping, crushing, shooting and burning (Jensen et al., 2001). J. Pharmacol. Although the ability to target rat ES cells was then available, doing so was still difficult. (B) Electronic von Frey (MouseMet, TopCat Metrology). Accordingly, dynamic weight bearing or gait analysis may provide similar data without the need for extensive animal and experimenter training. A disadvantage of this method is that the number of measurements per animal is variable and that it requires repeated, time-intensive measurements, which may lead to sensitization or learnt responses. Assessment of thermal sensitivity in rats using the thermal place preference test: description and application in the study of oxaliplatin-induced acute thermal hypersensitivity and inflammatory pain models. Neurosci. New hot plate tests to quantify antinociceptive and narcotic antagonist activities. Multimodal distribution of human cold pain thresholds. Data quality is usually improved if the time to occurrence of any behavior, rather than specific behavior types, is recorded, and if lower temperatures are used (Carter, 1991; Plone et al., 1996). Thus, the focus is often on reduction of the number of animals necessary to obtain data, and refinement of the method with the aim to decrease the amount of nociception caused to the animal. The larger size when compared to mice makes handling, sampling and performing procedures easier. A touch-on reaction is more likely to occur if the filament is not applied perpendicularly, if the filament is not applied smoothly, or if the filament moves horizontally during application, inducing scratching. According to the replacement principle, the use of live animals should be replaced with in vitro or computational methods where possible, and if unavoidable, the use of non-sentient or less sentient animals is preferred. Braz. Unfortunately, no accurate figures are available to determine precisely how many animals are used in experiments in the U.S. or worldwide. (2007). Cartil. Simmons is a student in the Kenan-Flagler Business School at the University of North Carolina at Chapel Hill. The MouseMet or RatMet electronic Von Frey systems deliver a mechanical stimulus via a hand-held probe (Figure 1B). Nat. The effects of morphine methadone and meperidine on some reflex responses of spinal animals to nociceptive stimulation. Most studies focus on either testing how a specific genetic pathway responds to the brain injury, or how administering a drug affects the severity of damage after injury. Third, aversive response to a cooling ramp can be used to determine the cold response threshold (Yalcin et al., 2009). (1991). A disadvantage of this method is that the number of measurements per animal is variable and that it requires repeated, time-intensive measurements, which may lead to sensitization or learnt responses. Med. Pain is a multifaceted and diverse experience that can be categorized into a number of types and modalities, depending on the presentation and triggering stimulus of the pain event. Assessment of pain and itch behavior in a mouse model of neurofibromatosis type 1. Allowing rodents to have several trial runs prior to the actual experiment can increase burrowing behaviors and reduce variability (Deacon, 2006b). Toxicol. Models and mechanisms of hyperalgesia and allodynia. doi: 10.1038/nrn2606, Moqrich, A., Hwang, S. W., Earley, T. J., Petrus, M. J., Murray, A. N., Spencer, K. S., et al. Pain 22, 131. doi: 10.1016/j.joca.2016.05.022, PubMed Abstract | CrossRef Full Text | Google Scholar, Allchorne, A. J., Broom, D. C., and Woolf, C. J. Methods 25, 241250. Sci. The distinction between nociception and pain thus underlines a key difference in terminology when referring to communicating and non-communicating subjects. doi: 10.1016/0304-3959(94)90023-x, Colburn, R. W., Lubin, M. L., Stone, D. J. Jr., Wang, Y., Lawrence, D., DAndrea, M. R., et al. Similar to the Hargreaves test, the thermal probe tests enables quantification of ipsilateral and contralateral heat thresholds in unrestrained mice, but with a shorter habituation time of 510 min. Pinpointing these should help researchers to develop rat genetic models of human disease. Some results suggest that the rat more accurately reflects human physiology than other species, mimicking human disease more accurately in many cases. Sci. Veterinarians are required to inspect the living conditions of the animals. A modified Hargreaves method for assessing threshold temperatures for heat nociception. This shift was primarily instigated by the availability of a much . Exceptionally adaptable, these rodents now inhabit almost all environments on Earth, especially near human settlements where they are often seen as pests. (2011). (C) Weight bearing (incapacitance test). Pain measurement: an overview. The behavioral methods used to measure nociception in rodents can be divided into stimulus-evoked (and further subdivided by the stimulus modalitymechanical, heat, cold) and non-stimulus evoked, with the most commonly used methods discussed in this review article. The dynamic weight-bearing test is able to detect reduced weight bearing behaviors of the affected hind limb in multiple pain models, including CFA-induced inflammation, chronic constriction injury, bone cancer pain and antigen-induced arthritis (Tetreault et al., 2011; Robinson et al., 2012; Quadros et al., 2015). Pharmacodyn. Front. A number of parameters are automatically analyzed by the software, including paw intensity, print area, stance phase duration (time spent on paw) and swing phase duration (time spent off paw). Front. Typically, noxious stimuli activate nociceptors, a subset of peripheral sensory neurons, which have a range of specialized ion channels and receptors that transduce noxious stimuli into electrical signals. While the underlying principle of the temperature gradient assay is similar to the two-temperature choice assay, the animal is free to explore along the gradient (usually between 4C and 65C over a length of 120 cm) until they settle within their preferred temperature or comfort zone. Analg. JRD, LSD and IV contributed to the preparation, revision and approval of the final manuscript. doi: 10.1016/s1474-4422(14)70024-9, Berge, O. G., Garcia-Cabrera, I., and Hole, K. (1988). Spontaneous burrowing behaviour in the rat is reduced by peripheral nerve injury or inflammation associated pain. Also, after advances in gene targeting and gene editing, it is possible to specifically modify the rat genome to produce knockouts and knockins, further enhancing our understanding of human disease through the development of powerful customized CRISPR rat models. Stimulus-evoked pain can be evaluated in humans using quantitative sensory testing. Similarly, human scoring lends itself to bias, although this can be avoided with appropriate randomization, allocation concealment and blind outcome assessment (Hirst et al., 2014). 47, 810. doi: 10.1016/s0304-3959(00)00276-1, Defrin, R., Ohry, A., Blumen, N., and Urca, G. (2002). In the case of researchers conducting surveys, for example, sample size is essential. Unequal weight distribution between the ipsilateral and contralateral paw are interpreted as a natural adjustment to the degree of nociception experienced, and have been observed in a number of models including monosodium iodoacetate (MIA)-induced osteoarthritis, bone cancer-induced pain, carrageenan-induced inflammation and sciatic nerve crush injury (Schtt et al., 1994; Medhurst et al., 2002; Bove et al., 2003; Buys and Alphonso, 2014). 130, 117122. Although animal research may have ethical concerns, the US has regulated its practice since 1966. Pain 59, 369376. A novel behavioral assay for measuring cold sensation in mice. A burrow filled with a suitable substrate (such as food pellets, sand, or marbles) is made from a long tube sealed at one end, secured and lifted by screws on the other end to prevent non-burrowing behaviors from displacing the substrate inside (Figure 6B). J. Neurosci. Advantages of the cold plate test are its relative speed and the ability for accurate temperature control. Attenuated cold sensitivity in TRPM8 null mice. Nevertheless, the acetone evaporation test has been validated in multiple models of inflammatory and neuropathic pain in both mice and rats (Choi et al., 1994; Colburn et al., 2007; Yamamoto et al., 2016). doi: 10.1016/s0165-0270(99)00004-7, Plone, M. A., Emerich, D. F., and Lindner, M. D. (1996). Research scientists worldwide agree that the use of rat models for disease research and experimentation has become more relevant in the past few years. Pain 9, 445452. In this experiment, a previously unafraid baby was conditioned to become afraid of a rat. Lett. Other advantages of this methodology include eliminating the phenomenon of clustered selection and a low . PLoS One 12:e0175839. Stimulus-evoked pain is described as either hyperalgesia or allodynia, and is further subdivided on the basis of the evoked stimulus modality (e.g., mechanical, heat, cold, chemical; Woolf and Mannion, 1999). In addition, a programmable hold step with constant force application can also be incorporated in the experimental setup to determine the time to withdrawal (Lu and Schmidtko, 2013). (B) Continuous temperature gradient assay. The Behavioral Spectrometer (Behavioral Instruments) is purpose built apparatus consisting of an enclosed box (~30 30 30 cm) with a ceiling-mounted fish-eye lens, accelerometer and a row of wall-mounted photobeams (Figure 6E). N Y Acad. Then in 2012, CRISPR/Cas9 changed genome manipulation as we know it. opportunities for species-specific behaviour), handling, procedures at daytime in nocturnal species, changes in groups for randomization, and too many male animals caged together. Cartil. doi: 10.1016/s0140-6736(99)01307-0. (2014). The time-to-integrate-to-nest test as an indicator of wellbeing in laboratory mice. The time taken to settle in a temperature zone and/or the temperature of the chosen zone is recorded. The criterion that constitutes a positive response to a filament varies between laboratories, with 20%40% withdrawal response rates over 510 applications being used typically (Scholz et al., 2005; Minett et al., 2011). Protoc. Brain Res. Phenotypic changes in diabetic neuropathy induced by a high-fat diet in diabetic C57BL/6 mice. The up-down method is practically limited by commercial filaments that are not equally spaced (therefore, the average increment is used for d), incorrect labeling of the force in log units, and the need for the first filament to be close to the mean threshold (which may be unknown; Bradman et al., 2015). 14 Articles, This article is part of the Research Topic, Creative Commons Attribution License (CC BY). Quantitative assessment of tactile allodynia in the rat paw. doi: 10.1080/23328940.2015.1135689, Vazquez, G., Alvarez, E., and Navaza, J. M. (1995). J. Pharmacol. A monofilament is applied perpendicularly to the plantar surface of the hind paw until it buckles, delivering a constant pre-determined force (typically 0.213.7 mN for mice and 5.998 mN for rats) for 25 s (Figure 1A). doi: 10.1016/j.neuron.2011.02.051, Wallace, V. C., Norbury, T. A., and Rice, A. S. (2005). Using the Mouse Grimace Scale to reevaluate the efficacy of postoperative analgesics in laboratory mice. Physiol. Non-response occurs when some subjects do not have the opportunity to participate in the survey. (2012). 10, 283294. Oxaliplatin administration increases expression of the voltage-dependent calcium channel 2-1 subunit in the rat spinal cord. J. Pharmacol. Rodents are placed individually in small enclosures with a glass floor. (D) Thermal probe test. Methods 53, 5563. Unlike the tail flick test, the hot plate test and other tests that apply heat stimuli to the hind paws are considered to integrate supraspinal pathways, as rats with spinal transection do not withdraw the hind limbs in the hot plate test (Giglio et al., 2006). 9, 807819. A major advantage of the thermal probe test is the reduced time required for acclimatization to the testing environment, enabling characterization of models or compounds with short duration of action, as well as testing of mechanical and thermal thresholds in the same enclosure. Alternatively, a continuous temperature gradienteither in linear (Figure 5B) or circular formcan be used to determine the preferred temperature in freely moving animals (Moqrich et al., 2005; Touska et al., 2016). While such behavioral changes may correlate to thermal allodynia or hyperalgesia, the contribution of additional sensations or complex behaviors to the preferred environmental temperature cannot be ruled out. It was around this time that gene editing came into being, specifically Zn Finger nucleases and TALENS. Computerized analysis of audible and ultrasonic vocalizations of rats as a standardized measure of pain-related behavior. Brain 125, 501510. Under the direction of the researcher, the apparatus applies a von Frey (0.5 mm) filament to the plantar surface, increasing the force incrementally (050 g) until the paw withdrawal threshold is reached. Mol. Meloxicam prevents COX-2-mediated post-surgical inflammation but not pain following laparotomy in mice. Woolf, C. J., and Mannion, R. J. The mouse has many similarities to humans in terms of anatomy, physiology and genetics. 95% of all animals used in research are rats because their genetic, biological and behavioral characteristics are so similar to that of humans. White, V.M.D., M.S., DACLAM, Dipl. In contrast to CPN, which is more common in males, nephrocalcinosis is more common in female rats. J. Neurosci. doi: 10.1016/0304-3959(94)90090-6, Carter, R. B. doi: 10.1093/gerona/glr210, OBrien, D. E., Brenner, D. S., Gutmann, D. H., and Gereau, R. W. IV (2013). doi: 10.1590/s0100-879x2006001200013, Gregory, N. S., Harris, A. L., Robinson, C. R., Dougherty, P. M., Fuchs, P. N., and Sluka, K. A. A rat model of bone cancer pain. While species such as saccharomyces cerevisiae, fruit flies, and zebrafish have been used for the study of cancer, organ development, recombination or large scale mutations, the rat model offers insights into human disease that the study of other species will not allow. Analysis of behaviors in unrestrained animals using automated technologies is increasingly being used to study non-stimulus evoked pain in rodents. TRPM8 mechanism of cold allodynia after chronic nerve injury. It is now possible to more easily manipulate the rat genome to produce specific gene knockouts and knockins and apply results to our understanding of human genetic disorders. This work was supported by an Australian Research Council Future Fellowship (IV). 119, 481488. Disadvantage 2: Uncoverage Bias A small sample size also affects the reliability of a survey's results because it leads to a higher variability, which may lead to bias. doi: 10.1371/journal.pone.0125822, Lu, R., and Schmidtko, A. The test is carried out on mesh floor and acetone is dabbed or sprayed on the plantar surface of the hind paw (Figure 4A), eliciting cooling of the skin to innocuous temperatures of 1521C (Colburn et al., 2007; Leith et al., 2010), although the actual temperature varies with ambient temperature, skin temperature, and the amount of acetone applied. Methods 31, 7983. Rolke, R., Baron, R., Maier, C., Tlle, T. R., Treede, R. D., Beyer, A., et al. Stimulus-evoked methods, which include manual and electronic von Frey, Randall-Selitto and the Hargreaves test, were the first to be developed and continue to be in widespread use. Most of the animals needed for medical and scientific inquiry in the U.S. are rodents (for example, rats and mice), and they are specifically bred for this purpose. (A) Tail flick test (radiant heat). 1, 383412. Neuropathic pain: diagnosis, pathophysiological mechanisms, and treatment. Neuro Oncol. J. Vis. doi: 10.1016/S0014-2999(01)01302-4, Jensen, T. S., and Yaksh, T. L. (1986). A disadvantage of systems that only use video recordings (e.g., DigiGait, GaitScan/TreadScan) is that they cannot measure paw print intensity or pressure (weight bearing parameters), which is relevant for pain models. With a much less expensive and more precise method for targeting genomes via embryos, the potential for rats as a model system of human disease could be pursued even easier. doi: 10.1016/S0304-3959(01)00437-7, Menndez, L., Lastra, A., Hidalgo, A., and Baamonde, A. Z., Rodriguez, K. B., Gallo, M. S., Pritchett-Corning, K., Karas, R. H., et al. Colver A, Fairhurst C, Pharoah PO. One way to evaluate nociception in non-communicating subjects is through observation of behavior. Facial expressions of mice can be used to score the subjective intensity of pain. Pain 5:18. doi: 10.1186/1744-8069-5-18, Hirst, J. Quantitative sensory testing in the german research network on neuropathic pain (DFNS): standardized protocol and reference values. The time taken to flick or withdraw the tail from the heat stimulus is recorded. Assoc. 101, 132143. 10:284. doi: 10.3389/fnmol.2017.00284. Animal models of pain: progress and challenges. Non-response occurs when some subjects do not have the opportunity to participate in the survey. doi: 10.1080/23328940.2016.1157668, Deuis, J. R., Whately, E., Brust, A., Inserra, M. C., Asvadi, N. H., Lewis, R. J., et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY).