The Hargreaves test, first described in 1988, is a method used to quantify heat thresholds in the hind paws of mice and rats upon application of a radiant or infrared heat stimulus (Hargreaves et al., 1988). To conduct a survey properly, you need to determine your sample group. ACS Chem. In cases of sensory loss, hypoalgesia may be present, which is defined as decreased sensitivity to a nociceptive stimulus. The software records the duration of different behavior types, including movement, grooming and rearing behaviors. Surface tension of alchol water + water from 20 to 50 degrees celcius. - 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. The rat grimace scale: a partially automated method for quantifying pain in the laboratory rat via facial expressions. The three Rs are: Reduction, Refinement, Replacement.. (2012). A camera below records the paw prints, which are illuminated by internally reflected light in the glass. In addition, skin and ambient temperature, the location of stimulus application on the tail, as well as learnt avoidance behaviors can affect the withdrawal response (Yoburn et al., 1984; Berge et al., 1988). Eur. A radiant or infrared heat source is focused on the plantar surface of the hind paw and the time taken to withdraw from the heat stimulus is recorded. 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. (A) Manual Von Frey. To address this, a modified Hargreaves test has been reported, that utilizes a feedback-controlled radiant heat source to apply a constant temperature to the hind paw. doi: 10.1016/0304-3959(92)90041-9, Knowlton, W. M., Bifolck-Fisher, A., Bautista, D. M., and McKemy, D. D. (2010). (E) Behavioral Spectrometer (Behavioral Instruments). Behaviors that are analyzed include locomotive activity (still, walking, trotting, running), distance traveled, velocity, grooming, posture, eating/drinking and foraging. An additional confounding factor in the hot plate test is the tendency for learned behavioral responses, which lead to diminished reaction times during subsequent exposures to the hot plate (Gamble and Milne, 1989; Plone et al., 1996). Nat. Activation of opioid receptors in cutaneous nerve endings by conorphin-1, a novel subtype-selective conopeptide, does not mediate peripheral analgesia. Neuron 70, 482494. J. This continues until at least four readings are obtained after the first change of direction. This book provides detailed information on a number of methodologies that can be used in The Rat: A Model Used in Biomedical Research Analysis of behaviors in unrestrained animals using automated technologies is increasingly being used to study non-stimulus evoked pain in rodents. Thus, pain is an experience that encompasses both sensory and emotional components; therefore the term pain is not interchangeable with nociception. Rodents are restrained and a focused beam of light is applied to tail. Mouse Biol. If a stimulus is applied that does not normally evoke a withdrawal response, and the animal withdraws from the stimulus, the animal is considered to have allodynia. The sequenced genome will also unveil new targets for drug intervention. Life Sci. No use, distribution or reproduction is permitted which does not comply with these terms. Pharmacodyn. While the method carries the disadvantage that the rodent has to be restrained, the tail flick test is of very short duration so handling can be minimized easily. 49, 117120. A modified Hargreaves method for assessing threshold temperatures for heat nociception. PLoS One 12:e0175839. J. 10, 283294. In this test, animals are placed individually in small cages with a mesh or otherwise penetrable bottom. Hyperalgesia and allodynia: peripheral mechanisms. doi: 10.1016/j.jneumeth.2015.08.010, Brenner, D. S., Golden, J. P., and Gereau, R. W. IV (2012). Finally, whisker change refers to change in whisker position (may be backward, forward, or clumped together). Pharmacological characterisation of the highly NaV1.7 selective spider venom peptide Pn3a. 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. Multimodal distribution of human cold pain thresholds. An overview of animal models of pain: disease models and outcome measures. Rat models are therefore still widely used. Lett. Can quantitative sensory testing move us closer to mechanism-based pain management? Picking up rats by the tail is stressful and should be avoided . doi: 10.1016/j.neuron.2011.02.051, Wallace, V. C., Norbury, T. A., and Rice, A. S. (2005). Meloxicam prevents COX-2-mediated post-surgical inflammation but not pain following laparotomy in mice. 120, 37603772. Behav. 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). Both the mouse and rat grimace scale are limited by the need for extensive training of the observer and a degree of subjectivity, which could lead to variability. Similarly, the terms nociception and pain are often used interchangeably, although the term pain is rarely appropriate to use in reference to animal studies. TRPM8, but not TRPA1, is required for neural and behavioral responses to acute noxious cold temperatures and cold-mimetics in vivo. J. Neurosci. It's a little like tossing a rat into a vat of gasoline and then concluding that gasoline causes drowning. Burrhus Frederic Skinner, also known as B.F. Skinner, is considered the "father of Operant Conditioning.". Electronic Von Frey systems operate under similar principles as manual von Frey, except that a single, un-bending filament is applied with increasing force until a paw withdrawal response is elicited. 1. doi: 10.1089/neu.2010.1700, Scholz, J., Broom, D. C., Youn, D. H., Mills, C. D., Kohno, T., Suter, M. R., et al. When rodents are unwell, the amount of material removed from the burrow is decreased. J. Pharmacol. In contrast to other Von Frey setups, animals are housed in individual enclosures with bars, rather than mesh, to help maximize the surface area of the hind paw available for application of the filament. 52, 34703478. Exp. A wide variety of other species make up the remaining 4%, from . Closely related to the last point is the subject of digital addiction. Depending on the species and strain of rodent used, at least 12 different behaviors have been noted in the hot plate test, including sniffing, grooming, stamping of the legs, freezing, licking, leaning and jumping (Espejo and Mir, 1993). Z., Rodriguez, K. B., Gallo, M. S., Pritchett-Corning, K., Karas, R. H., et al. Any long-term research involves mice and rats because of this unique aspect to the research. Anesth. Pain 66, 265270. 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. The distinction between nociception and pain thus underlines a key difference in terminology when referring to communicating and non-communicating subjects. If no nocifensive behaviors are observed, the animal must be removed from the hot plate after pre-determined cut-off time to prevent tissue damage. In this test, monofilaments of varying forces (in this case 0.6, 1, 1.4, 2, 4 and 6 grams-force) are applied in ascending order an equal number of times (in this case five times) and the response to each trial is recorded. Well-established strains of rat are used to study a number of human diseases such as: obesity and diabetes cancer cardiovascular disease (including high blood pressure and heart failure). Quantitative sensory testing in the german research network on neuropathic pain (DFNS): standardized protocol and reference values. 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. (2012). The Hargreaves test is usually carried out using a glass bottom enclosure, which can be heated to minimize errors arising from heat sink effects. doi: 10.1093/ilar.43.4.244, Gabriel, A. F., Marcus, M. A., Walenkamp, G. H., and Joosten, E. A. 89% of scientists surveyed by the Pew Research Center were in favor of animal testing for scientific research. 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. Their premise is based on the presumption that because we share similar biological processes, and because animals are whole, complex organisms, data obtained from an animal model can be extrapolated and . Burrowing behavior as an indicator of post-laparotomy pain in mice. Exp. 37, 18351841. As the animal is unaware of the researcher and neither needs to be restrained or trained, this technique eliminates operator subjectivity and reduces animal stress. There are protections in place for the animals. It should be noted that behavioral assessment of animals in groups (even if blinded) is typically not sufficient, with a preferred method being measurements performed on animals in random order by an investigator blinded to the treatment group each animal has been allocated to. The Advanced Dynamic Weight Bearing apparatus (Bioseb) was developed as a modification from static weight bearing or incapacitance tests and computes weight bearing for each of the front and rear paws, weight ratio and paw surface area in freely moving animals (Griffioen et al., 2015). Biol. Guidelines for the design and statistical analysis of experiments using laboratory animals. Rats have since been used to answer a wide range of basic science questions related to common human diseases in the fields of physiology, immunology, pharmacology, toxicology, nutrition, behavior and learning. However, the contribution of supraspinal processing to the tail flick response depends at least in part on the heating slope and temperature, with stimuli that lead to more delayed withdrawal responses generally considered to involve higher central nervous system functions considered necessary to process pain (Jensen and Yaksh, 1986). DAmour, F. E., and Smith, D. L. (1941). The mouse with a human ear on its back may have been one of the more bizarre and visually unsettling experiments carried out on a rodent, but mice have been used for scientific experiments since. Development of the Rodent Face Finder, which captures stills of rodent faces that a researcher then scores according to the relevant scale, has automated some of the experimental process (Sotocinal et al., 2011) albeit development of automatic facial expression scoring will make this approach even more widely useful. doi: 10.1016/j.jbspin.2004.01.010, Cox, J. J., Reimann, F., Nicholas, A. K., Thornton, G., Roberts, E., Springell, K., et al. doi: 10.1002/9780470942390.mo110116, Mogil, J. S. (2009). Using the horse grimace scale (HGS) to assess pain associated with acute laminitis in horses (Equus caballus). Similarities in pain descriptions of four different ethnic-culture groups. Twitter Brain Res. 6, 17511758. The burrowing assay has been validated for models of post-surgical pain in mice and in models of peripheral nerve injury, osteoarthritis and inflammation induced by CFA in rats (Jirkof et al., 2010; Andrews et al., 2012; Bryden et al., 2015). The term nociception was coined by Charles Sherrington in the early 1900s to distinguish the sensation of paina result of central nervous system processingfrom the physiological phenomenon of the peripheral nervous system responding to potential harmful stimuli (Dubner, 1983; Coutaux et al., 2005). doi: 10.1080/23328940.2016.1157668, Deuis, J. R., Whately, E., Brust, A., Inserra, M. C., Asvadi, N. H., Lewis, R. J., et al. Static weight bearing or incapacitance assays measure the distribution of weight across the hind paws and typically involve placing the animal in an inclined holder forcing placement of the hind paws on two independent pressure sensors (Figure 6C). 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). doi: 10.1111/ejn.12643, Tetreault, P., Dansereau, M. A., Dor-Savard, L., Beaudet, N., and Sarret, P. (2011). Quantitative assessment of tactile allodynia in the rat paw. Unlike the hot plate test, the cold plate test is particularly useful for models where only one paw is affected or sensitized by the experimental compound (unilateral pain) as guarding of the affected limb can be easily achieved, and thus can be easily quantified. Analgesic effects of clinically used compounds in novel mouse models of polyneuropathy induced by oxaliplatin and cisplatin. Monofilaments of differing forces are applied perpendicularly to the hind paw. Assessment of pain and itch behavior in a mouse model of neurofibromatosis type 1. Blocking caspase activity prevents transsynaptic neuronal apoptosis and the loss of inhibition in lamina II of the dorsal horn after peripheral nerve injury. For example, in models of burn pain, chemotherapy-induced neuropathy, and intra-articular carrageenan, changes in mechanical allodynia as measured by Von Frey and changes in gait parameters do not always correlate (Gabriel et al., 2009; Boehmerle et al., 2014; Yin et al., 2016; Deuis et al., 2017b). If you post a survey on your kitchen cleaner website, then only a small number of people have access to or knowledge about your survey, and it is likely that those who do participate will do so because they feel strongly about the topic. An advantage of this assay is that the endpoint is objective and requires minimal experience by the investigator to perform. Exceptionally adaptable, these rodents now inhabit almost all environments on Earth, especially near human settlements where they are often seen as pests. Researchers are often advised to begin their studies using rat models in these areas, not only because they are a suitable model, but also due to the fact that rats have already helped scientists reach a few significant breakthroughs in these areas. At least six responses around the estimated threshold are required for optimal calculation of the 50% threshold (Dixon, 1980). The test begins by assessing the response to a filament estimated to be close to the 50% withdrawal threshold (in this case 1 grams-force). Neurosci. 13, 587599. Despite being considered an innocuous stimulus, nave mice and rats can have a nocifensive response to the application of acetone, likely due to the concurrent olfactory or auditory (spray) stimuli, reducing the sensitivity of the assay (Colburn et al., 2007; Yamamoto et al., 2016). doi: 10.1038/nrn2606, Moqrich, A., Hwang, S. W., Earley, T. J., Petrus, M. J., Murray, A. N., Spencer, K. S., et al. doi: 10.1371/journal.pone.0104458, Minett, M. S., Quick, K., and Wood, J. N. (2011). Efficacy of drugs with different mechanisms of action in relieving spontaneous pain at rest and during movement in a rat model of osteoarthritis. Lancet 353, 19591964. 97:e52640. 4:51. doi: 10.3389/fvets.2017.00051, Vissers, K., and Meert, T. (2005). (1988). J. 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). doi: 10.1016/j.neuron.2007.04.017, Coutaux, A., Adam, F., Willer, J. C., and Le Bars, D. (2005). Pain 22, 131. Guidelines for reporting experiments involving animals: the ARRIVE guidelines. Therefore, there is an urgent need to increase our understanding of the underlying mechanisms of pain and to develop new treatments. Methods 255, 92103. In fact, over 90% of test subjects are mice, birds and rats. Rodents are commonly used to study the pathophysiological mechanisms of pain as studies in humans may be difficult to perform and ethically limited. Some examples of human disorders and diseases for which mice and rats are used as models include: Hypertension Diabetes Cataracts Obesity Seizures Respiratory problems Deafness Parkinson's. doi: 10.1523/JNEUROSCI.0122-10.2010, Ltsch, J., Dimova, V., Lieb, I., Zimmermann, M., Oertel, B. G., and Ultsch, A. 95% of animals used in experiments are not protected by the federal Animal Welfare Act (AWA), which excludes birds, rats and mice bred for research, and cold-blooded animals such as reptiles and most fish. General adaptation syndrome was discovered accidentally by Selye whilst he was conducting experiments on rats at McGill University in Montreal, Canada. Alternatively, voluntary response bias occurs when only a small number of non-representative subjects have the opportunity to participate in the survey, usually because they are the only ones who know about it. 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. 34, 17551762. Neurosci. However mice are less likely to survive the hypoxia-ischemia procedure, display a wider variety of brain damage, and have dramatic strain-to-strain differences. Not only does your survey suffer due to timing, but the number of subjects does not help make up for this deficiency. February 8, 2018| Kristen Coughlin | CRISPR, Rat Models, Last Updated on January 23, 2023 by ingenious. Cerebral palsy. Pain 22, 130136. (2005). Gait analysis methods for rodent models of osteoarthritis. Woolf, C. J., and Mannion, R. J. Painful and painless channelopathies. doi: 10.1016/j.pain.2013.06.015, Keywords: pain, rodent, hyperalgesia, allodynia, spontaneous pain, mechanical, heat, cold, Citation: Deuis JR, Dvorakova LS and Vetter I (2017) Methods Used to Evaluate Pain Behaviors in Rodents. 1, 28282830. Researchers choose rats over other species for a number of reasons: Eur. Some of the disadvantages of choosing a different animal model for these fields could be difficulty in collecting accurate data, comparing data from previous studies that used rat models, and drawing definitive conclusions that could apply to future studies. Although animal research may have ethical concerns, the US has regulated its practice since 1966. It is a low-cost, single-use product made from sterilized quarry sand and treated with a hydrophobic coating. 111, 409419. Genetically engineered mouse models recapitulate aspects of the corresponding human diseases and are instrumental in studying disease mechanisms and testing therapeutic strategies. 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. Role of the NLRP3 inflammasome in a model of acute burn-induced pain. Facial expression is subjectively scored for severity of pain based on five facial features (ear position, eye closing, cheek bulging, whisker position, and nose bulging). Pain 16, 485495. doi: 10.1038/nprot.2006.171, Deacon, R. M. (2006b). In 2001 however with the development of targeted genetic manipulation in mouse embryonic stem (ES) cells, rats took somewhat of a back seat as the rodent of choice for specific gene targeting because this technology was not yet available in the rat. While validated using an incisional model of pain in rats, this method takes longer to perform than the normal Hargreaves test and is not available to purchase commercially. Mice are cost effective because they are cheap and easy to look after. J. Methods 31, 7983. If an individual is on a company's website, then it is likely that he supports the company; he may, for example, be looking for coupons or promotions from that manufacturer. 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. LabSand is a revolutionary option for the collection of small urine samples in laboratory and research animals. While several systems are commercially available, the Dynamic Plantar Aesthesiometer (Ugo Basile) and MouseMet or RatMet (TopCat Metrology) are particularly robust and user-friendly systems. However, the force required to trigger probe heating is low (~1 g), adjustable, and the surface of the heating probe is rather large compared to the punctate von Frey filaments. With the sequencing of the Brown Norway rat in 2004 by the Rat Genome Sequencing Project Consortium, led by the Baylor College of Medicine, it is now known that almost all disease-linked human genes have counterparts in the rat. The burrows are placed in the rodents cage for a pre-determined duration and the amount of material displaced is weighed and recorded (Deacon, 2006b; Jirkof et al., 2010). Sci. 47, 810. New hot plate tests to quantify antinociceptive and narcotic antagonist activities. Consistent and precise placement of the filament is important to reduce intra-subject and inter-subject variability, with the specific placement dependent on the innervation territories of the test area and the model used. Typically, the sensation of warm is elicited at temperatures of 3437C, while the sensation of pain is elicited at temperatures of 4248C (Pertovaara et al., 1996; Defrin et al., 2006; Rolke et al., 2006). A number of endpoints can be obtained from the cold plate test, similar to the hot plate test. In the percent response method, several Von Frey filaments of varying forces are applied in ascending order an equal number of times (usually 510 applications) regardless of response, and the number of positive responses to each filament is converted to a percent response (Kim and Chung, 1992; Chaplan et al., 1994; Figure 2C). (A) The up-down method. While rats tend the habituate quickly (<15 min), mice can take up to an hour or more to settle in the cage before testing can begin, which can be time consuming (Chaplan et al., 1994; Minett et al., 2011). Joint Bone Spine 72, 359371. Weight bearing as a measure of disease progression and efficacy of anti-inflammatory compounds in a model of monosodium iodoacetate-induced osteoarthritis. Limitations of the Animal Model. This problem can be overcome by restraining the rodent and only placing the plantar surface of a one hind paw on the metal surface and recording the time to withdrawal, however this method requires significant handling and associated stress (Menndez et al., 2002). Now, researchers no longer have to choose between the anatomical advantages of rats and the genetic tractability of mice, due to new methods for introducing genetic modifications into the rat genome. doi: 10.1016/j.joca.2015.05.001, Buys, M. J., and Alphonso, C. (2014). (2017b). 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. Static hyperalgesia can be superficial or deep and is assessed by the application of pressure to the skin or underlying tissue by a finger or using a pressure algometer (Jensen and Finnerup, 2014). 198, 477480. Williams, W. O., Riskin, D. K., and Mott, A. K. (2008). The force ramp and paw withdrawal force are displayed by the software post-test. The time taken to flick or withdraw the tail from the heat stimulus is recorded. Burrowing, a spontaneous and self-motivated behavior, can be used as a measure of spontaneous or non-stimulus evoked nociception in mice and rats. Pain 20, 231240. ECLAM Corporate VP, Veterinary and Professional Services BACKGROUND Laboratory rodents have been used in the United States as research animals since the late 1800s. doi: 10.1016/0024-3205(85)90227-9. FIGURE 2 Figure 2 .Visual representation of the different methodological approaches used to determine mechanical sensitivity using manual Von Frey. doi: 10.1016/0304-3959(91)90207-e, Chaplan, S. R., Bach, F. W., Pogrel, J. W., Chung, J. M., and Yaksh, T. L. (1994). The method is based on the application of monofilaments with increasing force until a withdrawal response is elicited, and the force of the von Frey filament that elicits this positive response is designated as the mechanical withdrawal threshold (Figure 2B). The menthol receptor TRPM8 is the principal detector of environmental cold. Then there is the Sprague Dawley, an outbred albino preferred for behavioral studies. Behavioral and physiological methods for early quantitative assessment of spinal cord injury and prognosis in rats. Mol. While the Randall-Selitto test often results in similar types of outcomes to the Von Frey filament tests (Santos-Nogueira et al., 2012), the mechanical stimulation differs fundamentally from Von Frey filaments, which may also activate low-threshold mechanoreceptors in addition to nociceptors. Adams, B. L., Guo, W., Gors, R. T., and Knopp, K. L. (2016). Pain 96, 129140. 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. Res.
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