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PLoS One 9:e104458. This book condenses information from many sources on topics related to the care and use of rats in research. doi: 10.1021/acschemneuro.5b00113, Deuis, J. R., Zimmermann, K., Romanovsky, A. Eur. Research scientists worldwide agree that the use of rat models for disease research and experimentation has become more relevant in the past few years. 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. One area where rats have contributed to major insights is the field of cerebral palsy research. This work was supported by an Australian Research Council Future Fellowship (IV). 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. 2011:848307. doi: 10.1155/2011/848307, Hager, C., Biernot, S., Buettner, M., Glage, S., Keubler, L. M., Held, N., et al. Nonetheless, consistent measurements are possible in acclimatized animals, although guarding or altered weight distribution may lead to errors. 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). A single, un-bending filament is applied perpendicularly to the hind paw. doi: 10.1097/fbp.0000000000000026, Banik, R. K., and Kabadi, R. A. Robinson, I., Sargent, B., and Hatcher, J. P. (2012). Lancet 353, 19591964. Neuropathic pain: aetiology, symptoms, mechanisms, and management. Pain 59, 8594. The hot plate test, first described in 1944, can be used to determine heat thresholds in mice and rats (Woolfe and Macdonald, 1944). Thus, the hot plate test can produce greatly variable data, even within laboratories. Pain 68, 133140. Hyperalgesia and allodynia: peripheral mechanisms. Exposure of the hind paw to acetone does not evoke paw withdrawal in lightly anesthetized animals (unlike the tail flick assay), while ethyl chloride application achieves skin temperatures approaching 5C or less, and is generally considered a noxious cold stimulus (Leith et al., 2010). Osteoarthr. Rodents are allowed to freely move along a liner or circular surface with a temperature gradient. doi: 10.1213/ane.0000000000000291, Carlton, S. M., Lekan, H. A., Kim, S. H., and Chung, J. M. (1994). As altered weight bearing can be a major symptom of human pain conditions, these tests were developed to improve translation of rodent nociception models to the clinic. The assay is also capable of detecting side effects of analgesics, such as drowsiness, although it may be difficult to distinguish whether decreased burrowing arises from lack of efficacy or adverse effects unless additional behavioral tests or full dose-response curves are performed (Andrews et al., 2012). Electrophysiological, behavioral and histological characterization of paclitaxel, cisplatin, vincristine and bortezomib-induced neuropathy in C57Bl/6 mice. 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). Eur. Protoc. This sample group should include individuals who are relevant to the survey's topic. An additional disadvantage of incapacitance or static weight bearing tests is that only models with unilateral hind paw nociception can be assessed in this manner. Diabetes Res. doi: 10.1016/S1474-4422(10)70143-5, Bautista, D. M., Siemens, J., Glazer, J. M., Tsuruda, P. R., Basbaum, A. I., Stucky, C. L., et al. As the nocifensive response can be too fast for an investigator to quantify in real time, video recordings that are played back in slow motion may be required to accurately analyze the response to acetone. Efficient analysis of experimental observations. A response is considered positive if the animal exhibits any nocifensive behaviors, including brisk paw withdrawal, licking, or shaking of the paw, either during application of the stimulus or immediately after the filament is removed. 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). Guidelines for the design and statistical analysis of experiments using laboratory animals. 49, 117120. 198, 477480. Anim. doi: 10.1097/01.ajp.0000154048.68273.d8, Dell, R. B., Holleran, S., and Ramakrishnan, R. (2002). Using animals in experiments | The Humane Society of the United States Brain Res. 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). Whether you need help solving quadratic equations, inspiration for the upcoming science fair or the latest update on a major storm, Sciencing is here to help. doi: 10.1038/nature05910, Bennett, D. L., and Woods, C. G. (2014). The influence of immaturity on hypoxic-ischemic brain damage in the rat. 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. TRPM8, but not TRPA1, is required for neural and behavioral responses to acute noxious cold temperatures and cold-mimetics in vivo. (2016). Neurosci. Oxaliplatin administration increases expression of the voltage-dependent calcium channel 2-1 subunit in the rat spinal cord. Three main ethical attitudes towards animals are reviewed here. For detailed protocols of these methods see Minett et al. The principles that apply to mice generally apply to rats and other laboratory rodents, but species-specific differences in behaviour and size alter the specifics of handling procedures that work well in each species. Accordingly, translation of research using experimental nociceptive assays to pain treatment in the clinic has met with some difficulties. (2013). 160, 15731576. In practice, this test is useful for assessment of nociceptive thresholds in rats rather than mice as animals need to be heavily physically restrained with the tested paw held out, and mice rarely tolerate such handling (Anseloni et al., 2003; Minett et al., 2011; Santos-Nogueira et al., 2012). 32, 5260. While the underlying protocols and principles are different, it remains to be determined if electronic Von Frey activates a different subset of sensory neurons compared to manual Von Frey (e.g., high-threshold vs. low threshold mechanoreceptors). doi: 10.1016/s1063-4584(03)00163-8, Bradman, M. J., Ferrini, F., Salio, C., and Merighi, A. 67, 705713. Their brains are larger than mice, and the animals are less timid and more intelligent. (1974). Differentiating thermal allodynia and hyperalgesia using dynamic hot and cold plate in rodents. 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. Technology Addiction. This book provides detailed information on a number of methodologies that can be used in The Rat: A Model Used in Biomedical Research Pharmacodyn. In addition to burrowing, other spontaneous behaviors can be assessed, including nesting construction and food hoarding (Deacon, 2006a; Rock et al., 2014). Clin. doi: 10.1371/journal.pone.0098856, Huang, S. M., Li, X., Yu, Y., Wang, J., and Caterina, M. J. 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. The sensation of pleasant or innocuous cooling is typically elicited at temperatures of ~2329C, while the sensation of cold pain is significantly variable, with multimodal distribution of the cold pain threshold recently reported, corresponding to modal threshold temperatures of 23.7C, 13.2C and 1.5C, respectively (Ltsch et al., 2015). Nocifensive behaviors include forepaw withdrawal or licking, hind paw withdrawal or licking, stamping, leaning posture and jumping (Espejo and Mir, 1993). Voluntary response bias is another disadvantage that comes with a small sample size. But the missing genes signal a problem for using gene editing to de-extinct species. (2015). 14 Pros and Cons of Animal Research - Vittana.org PLoS One 9:e98856. Life Sci. Sci. (2003). doi: 10.1016/s0165-0270(01)00483-6, Millecamps, M., Jourdan, D., Leger, S., Etienne, M., Eschalier, A., and Ardid, D. (2005). Nature 444, 894898. 52, 34703478. In its simplest form, the animal can choose between two adjacent areas maintained at different temperatures. Burn Care Res. doi: 10.1016/0006-8993(86)91138-8, Jirkof, P., Cesarovic, N., Rettich, A., Nicholls, F., Seifert, B., and Arras, M. (2010). 9, 8998. PLoS One 7:e39765. Automated behavioral analysis can be performed in a dedicated apparatus (Behavioral Spectrometer, Behavioral Instruments) or in a home cage (HomeCageScan, CleverSys; PhenoTyper, Noldus), using automated video analysis, vibration sensors, photobeams, and combinations thereof. 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. 1, 383412. The Disadvantages of a Small Sample Size | Sciencing Neurosci. Characterization of the foot withdrawal response to noxious radiant heat in the rat. (1988). Digigait quantitation of gait dynamics in rat rheumatoid arthritis model. Anim. 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). Pain 56, 155166. Rumajogee P, Bregman T, Miller SP, Yager JY, Fehlings MG. 2016. A behavioral and pharmacological validation of the acetone spray test in gerbils with a chronic constriction injury. (B) Cold plantar assay. Readership. (2017). Most importantly, however, from the very beginning their knowledge and competence convinced me they would deliver on our project, so I trusted them and they fully met our expectations. doi: 10.1152/physrev.00025.2008, Santos-Nogueira, E., Redondo Castro, E., Mancuso, R., and Navarro, X. Both versions of the test require the animal to be loosely restrained. However, concerns over the clinical translatability of stimulus-evoked nociception in recent years has led to the development and increasing implementation of non-stimulus evoked methods, such as grimace scales, burrowing, weight bearing and gait analysis. Then in 2012, CRISPR/Cas9 changed genome manipulation as we know it. Animals 6:47. doi: 10.3390/ani6080047. (B) Continuous temperature gradient assay. However, replacement or substitution of animals for nonsentient materials is difficult in pain research due to the nature of the behavioral experiments. Differentiating analgesic and non-analgesic drug activities on rat hot plate: effect of behavioral endpoint. doi: 10.1523/JNEUROSCI.0122-10.2010, Ltsch, J., Dimova, V., Lieb, I., Zimmermann, M., Oertel, B. G., and Ultsch, A. Variability is determined by the standard deviation of the population; the standard deviation of a sample is how the far the true results of the survey might be from the results of the sample that you collected. This review article will provide an overview of the current behavioral methods that are used to assess pain behaviors in mice and rats. The time spent on the test plate relative to the reference plate is measured over a set period of time. Pain cannot be directly measured in animals; instead pain is inferred from pain-like behaviors, such as withdrawal from a nociceptive stimulus, which is the most commonly used method to quantify nociception in animal studies. Rock, M. L., Karas, A. Methods Mol. 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). However, it is unclear to what degree changes in gait in rodent models reflect altered pain or nociception, or conversely, anti-nociception or analgesia. Randall-Selitto test: a new approach for the detection of neuropathic pain after spinal cord injury. Acetylsalicylic acid, paracetamol and morphine inhibit behavioral responses to intrathecally administered substance P or capsaicin. (2014). Abstract The laboratory rat was the first mammal domesticated for research purposes. Nat. Lab. It is plausible that differences in behavior may relate to the type of sensory fiber activated. For instance, improving data homogeneity and enhancing statistical power (Dell et al., 2002) will result in fewer animals being necessary to achieve the required confidence level (Festing and Altman, 2002). Brain 125, 501510. The U.S. Department of Agriculture (USDA) does compile annual statistics on some animals used in . Thus, the term nociception is used to describe the peripheral neuronal response to noxious stimuli, which encompasses any stimuli, being mechanical, thermal, electrical or chemical, that have the potential to damage are damaging to tissue (Dubin and Patapoutian, 2010). In the conventional hot plate test the rodent is placed on a metal surface maintained at a constant temperature (in this case 54C) and the time taken to elicit a nocifensive behavior (e.g., hind paw withdrawal or licking) is recorded. Second, the number of flinches over a set period of time can be recorded at a specific temperature (Deuis et al., 2013; Zimmermann et al., 2013). Xing, H., Chen, M., Ling, J., Tan, W., and Gu, J. G. (2007). These techniques provide an insight into how sensitive to cold temperatures the animal is, and thus provides an indirect measure of cold-induced hyperalgesia and allodynia. 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. doi: 10.1111/j.1476-5381.2010.00873.x, Medhurst, S. J., Walker, K., Bowes, M., Kidd, B. L., Glatt, M., Muller, M., et al. (2007). This therefore provides measure of paw withdrawal threshold on a continual scale, as the force is applied continuously and not in steps. J. Med. J. Clin. In the past, housing of rodents used for research was designed primarily to prevent Ann. Its characterized primarily by motor impairment, but there are also a range of accompanying conditions such as seizure disorders and cognitive defects. This review article provides an overview, as well as discussion of the advantages and disadvantages of the most commonly used behavioral methods of stimulus-evoked and non-stimulus-evoked nociception used in rodents. 43, 207213. (A) Acetone evaporation test. Anim. 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. 9, 807819. Prior to the development of automated digitized platforms, this test was performed by coloring the animals paws with ink; the animal was then allowed to walk freely on a paper, which could be scanned for analysis (Ishikawa et al., 2014). (2013). doi: 10.1371/journal.pone.0125822, Lu, R., and Schmidtko, A. 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. Lett. doi: 10.1038/nprot.2006.19, Decosterd, I., and Woolf, C. J. DAmour, F. E., and Smith, D. L. (1941). Neurosci. Mol. Exp. Analgesic effects of clinically used compounds in novel mouse models of polyneuropathy induced by oxaliplatin and cisplatin. Third, aversive response to a cooling ramp can be used to determine the cold response threshold (Yalcin et al., 2009). (B) The ascending stimulus method. doi: 10.1016/j.pain.2013.05.032, Dixon, W. J. 14:14. doi: 10.1186/1471-2474-14-14, Pertovaara, A., Kauppila, T., and Hmlinen, M. M. (1996). However, with the use of animal models come challenges relating to the appropriate quantification of behavioral responses that could be considered equivalent to pain in humans. This includes minimizing the duration of models, replacing nocifensive model compounds for ones that cause shorter lasting nociception, or reducing the administered doses of compounds. LabSand is a revolutionary option for the collection of small urine samples in laboratory and research animals. 27, 14. Behav. Click the button below to get a quote! Experimenters experienced in the technique are able to distinguish between touch on or false positive responses, however, this can be difficult for inexperienced researchers and extensive training is usually required to produce high quality data. The same is likely to be true of other rodent species, many of . J. Pharmacol. Am. Neurosci. Lab. To evaluate thermal sensitivity, the time taken to settle in a temperature zone, as well as the temperature of the chosen zone, can be compared to control animals. General Adaptation Syndrome - Psychologist World (C) The percent response method. (2012). The intensity of the light source should be adjusted to produce withdrawal latencies of 1012 s in nave animals, providing a sufficient window to detect heat allodynia and hypoalgesia, with a pre-determined cut off time to prevent tissue damage. Pain 50, 355363. 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. 53, 2428. Methods 25, 241250. The Practical Mouse Despite the advantages rats have over mice as human analogs, mice have dominated the modern genetic and biomedical research landscape. doi: 10.1016/s0304-3959(96)03177-6, Yin, K., Deuis, J. R., Lewis, R. J., and Vetter, I. BMC Musculoskelet. Figure 5.Temperature preference assays. Pain measurement: an overview. Disord. The Hargreaves test permits measurement of ipsilateral and contralateral heat thresholds, allowing each animal to serve as its own internal control in unilateral pain models. A number of endpoints can be obtained from the cold plate test, similar to the hot plate test. The rodent is placed in an inclined holder with the hind paws resting on two separate pressure sensors. A large number of parameters can be analyzed, including paw intensity (a measure of paw pressure or weight bearing), paw print parameters (e.g., toe spread, print length, print width, print area), dynamic parameters (e.g., stance phase, swing phase, duty cycle, stride length, swing speed) and regularity index (a measure of interlimb coordination). An animal model of oxaliplatin-induced cold allodynia reveals a crucial role for Nav1.6 in peripheral pain pathways. Twitter Pain 20, 231240. Rodents are placed individually in small cages with a mesh or barred floor. doi: 10.1016/0160-5402(91)90014-v, Touska, F., Winter, Z., Mueller, A., Vlachova, V., Larsen, J., and Zimmermann, K. (2016). In addition, welfare benefits in form of testing of unrestrained mice and exposure of only a single hind paw to a noxious heat stimulus are favorable. doi: 10.1016/s0165-0270(03)00241-3, Balayssac, D., Ling, B., Ferrier, J., Pereira, B., Eschalier, A., and Authier, N. (2014). Neuron 54, 379386. 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). 96, 312317. In line with this, the uptake of grimace scales to study pain in the scientific community has been low, with approximately 30 studies published using the method in rodents since its introduction by Langford et al. doi: 10.1016/j.bbr.2008.12.018, Gamble, G. D., and Milne, R. J. 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. 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). A modified Hargreaves method for assessing threshold temperatures for heat nociception. As a result, mice became the more prominent model organism due to the availability of genetically modified lines where a gene of interest can be activated or inactivated in specific cells. Sci. 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. Pain 1:36. doi: 10.1186/1744-8069-1-36, Andrews, N., Legg, E., Lisak, D., Issop, Y., Richardson, D., Harper, S., et al. Different methodological approaches are used to determine mechanical sensitivity using manual Von Frey, including the up-down, ascending stimulus or percent response method, all of which will be discussed below. Thus, pain is an experience that encompasses both sensory and emotional components; therefore the term pain is not interchangeable with nociception. Nurs. Temperature preference assays are typically relatively fast and require little rodent handling or restraint (Morgan et al., 2012). 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). Pain 87, 149158. This research has almost exclusively used a single procedure to induce the condition in animal models. Allowing rodents to have several trial runs prior to the actual experiment can increase burrowing behaviors and reduce variability (Deacon, 2006b). Although the ability to target rat ES cells was then available, doing so was still difficult. The latency to paw withdrawal is recorded and used to quantify cold allodynia and hyperalgesia. 1020, 215221. Ultrasound vocalisation by rodents does not correlate with behavioural measures of persistent pain. Burrowing behavior as an indicator of post-laparotomy pain in mice. A cold stimulus is delivered by applying a cut off syringe filled with dry ice (for temperature ranges of 512C) or wet ice (temperature of 17C) to the glass underneath the paw (Brenner et al., 2012, 2015; Figure 4B). In fact, it is a requirement for publication of in vivo data in high quality journals that relevant standards and guidelines are strictly adhered to (McGrath et al., 2010). Irwin, S., Houde, R. W., Bennett, D. R., Hendershot, L. C., and Seevers, M. H. (1951). A similar scale has also been developed for rats. doi: 10.1016/0304-3959(92)90041-9, Knowlton, W. M., Bifolck-Fisher, A., Bautista, D. M., and McKemy, D. D. (2010). doi: 10.1371/journal.pone.0039765, Brenner, D. S., Golden, J. P., Vogt, S. K., and Gereau, R. W. (2015). Mice and Rats as Laboratory Animals - MSD Veterinary Manual The thermal probe test (MouseMet Thermal, Topcat Metrology) is a novel method recently described to quantify heat thresholds in mice (Deuis and Vetter, 2016). Using the mouse grimace scale to assess pain associated with routine ear notching and the effect of analgesia in laboratory mice. Age-related neurodegenerative diseases are largely limited to humans and rarely occur spontaneously in animals. The clinically translatability of the tail flick test is therefore unclear. These measures can have a profound effect on the apparent anti-nociceptive efficacy of test compounds and should thus be carefully considered during experimental design (Winter and Flataker, 1965).
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