Allodynia and hyperalgesia in neuropathic pain: clinical manifestations and mechanisms. Pain research in animals. Rodent models of cerebral palsy have been studied for almost forty years to fill the gaps in our knowledge, hopefully pointing the way to future treatments. Burrhus Frederic Skinner, also known as B.F. Skinner, is considered the "father of Operant Conditioning.". Accordingly, translation of research using experimental nociceptive assays to pain treatment in the clinic has met with some difficulties. Researchers choose rats over other species for a number of reasons: 9, 8998. doi: 10.1523/JNEUROSCI.0122-10.2010, Ltsch, J., Dimova, V., Lieb, I., Zimmermann, M., Oertel, B. G., and Ultsch, A. Am. Behaviors that are analyzed include locomotive activity (still, walking, trotting, running), distance traveled, velocity, grooming, posture, eating/drinking and foraging. Eur. While not in routine clinical use, quantitative sensory testing has the potential to improve patient outcomes by classifying pain based on the mechanism and choosing treatments that target that mechanism (Baron et al., 2010; Cruz-Almeida and Fillingim, 2014). 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. 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. Pain 3:40. doi: 10.1186/1744-8069-3-40, Pitcher, G. M., Ritchie, J., and Henry, J. L. (1999). 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. 13, 587599. (2014). 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. Neuropharmacology 23, 915924. 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. Click the button below to get a quote! Pain 9, 445452. Consistent application of acetone can be challenging, as acetone has a lower surface tension than water (25.2 mN/m and 72.8 mN/m respectively), making it difficult to form uniform drops with a pipette or syringe (Vazquez et al., 1995), with some laboratories opting to use a spray instead (Yamamoto et al., 2016). J. In the past, housing of rodents used for research was designed primarily to prevent Assoc. 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 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). Received: 04 April 2017; Accepted: 22 August 2017; Published: 06 September 2017. Curr. doi: 10.1016/j.ejpain.2011.07.012, Ankier, S. I. Deacon, R. M. (2006a). It should be noted that no test can therefore measure pain in animals directlythe presumably unpleasant emotional experience of pain is inferred from pain-like behaviors which can include the withdrawal of a body part from a stimulus, reduced ambulation, agitation, an increase in grooming of the affected area, and vocalizations upon sensory stimulation. Pain 87, 149158. The main advantage of the thermal probe test is that the mice are placed in individual runs standing on bars instead of glass enabling access to the plantar surface, which allows simultaneous assessment of mechanical thresholds by von Frey, removing the need for acclimation in two different enclosures. Analg. The time-to-integrate-to-nest test as an indicator of wellbeing in laboratory mice. 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. It is the premier source of information on the laboratory rat. Research scientists worldwide agree that the use of rat models for disease research and experimentation has become more relevant in the past few years. Electrophysiological, behavioral and histological characterization of paclitaxel, cisplatin, vincristine and bortezomib-induced neuropathy in C57Bl/6 mice. doi: 10.1093/ilar.43.4.207, Deuis, J. R., Dekan, Z., Wingerd, J. S., Smith, J. J., Munasinghe, N. R., Bhola, R. F., et al. 25, 99111. doi: 10.1523/JNEUROSCI.1526-05.2005, Schtt, E., Berge, O. G., Angeby-Mller, K., Hammarstrm, G., Dalsgaard, C. J., and Brodin, E. (1994). Oxaliplatin administration increases expression of the voltage-dependent calcium channel 2-1 subunit in the rat spinal cord. Facial expressions of mice can be used to score the subjective intensity of pain. These neurons are pseudo-unipolar, with a peripheral branch that terminates in the skin or viscera and a central branch that terminates in the spinal cord. Pain measurement: an overview. (2016). Animals are stressed - there are many reasons for stress in the animals: transportation, insufficient time for adapting, cages without enrichment (e.g. Osteoarthr. The rodent is placed in an inclined holder with the hind paws resting on two separate pressure sensors. The influence of immaturity on hypoxic-ischemic brain damage in the rat. Nevertheless, the temperature preference test has been used extensively in the study of the role of thermosensitive transient receptor potential (TRP) channels in thermal nociception including TRPA1, TRPM8, TRPM3, TRPV3 and TRPV4 (Bautista et al., 2007; Knowlton et al., 2010; Huang et al., 2011; Vriens et al., 2011; Touska et al., 2016). Methods 87, 185193. However, theyre more widely used in studying cardiovascular and metabolic disorders, as well as the onset of conditions that relate to the brain, digestive system, and various health problems related to behavior. The framework for these standards and guidelines are based on the principles of the 3Rs (replacement, reduction, refinement). Differentiating thermal allodynia and hyperalgesia using dynamic hot and cold plate in rodents. Cerebral palsy. Pain 154, 19992006. 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. She specializes in business, consumer products, home economics and sports and recreation. However, this also requires animals to be acclimatized to the cages, to ensure ambulation and exploratory behaviors, which could be misinterpreted as a positive response, are kept to a minimum. Rep. 7:40883. doi: 10.1038/srep40883, Deuis, J. R., Yin, K., Cooper, M. A., Schroder, K., and Vetter, I. Rat models are therefore still widely used. Undoubtedly, nociception, or the ability to detect a potentially harmful stimulus, is a fundamental physiological function in mammals and indeed many other species. 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. ILAR J. The hot plate test, first described in 1944, can be used to determine heat thresholds in mice and rats (Woolfe and Macdonald, 1944). Surface tension of alchol water + water from 20 to 50 degrees celcius. The Little Albert Experiment demonstrated that classical conditioning could be used to create a phobia. Methods Mol. The rodent is restrained and the hind paw (or tail) is placed between a pointed probe tip and flat surface. Sci. ECLAM Corporate VP, Veterinary and Professional Services BACKGROUND Laboratory rodents have been used in the United States as research animals since the late 1800s. Automated analysis of postoperative behaviour: assessment of HomeCageScan as a novel method to rapidly identify pain and analgesic effects in mice. 11, 821830. Rodents are commonly used to study the pathophysiological mechanisms of pain as studies in humans may be difficult to perform and ethically limited. (2006). 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. The dynamic hot plate test, first described in 1984, uses an increasing temperature ramp rather than a constant temperature. The temperature preference is used as a surrogate measure of thermal aversion and aims to assess temperature preference in rodents. The thermal probe test (MouseMet Thermal, Topcat Metrology) is a novel method recently described to quantify heat thresholds in mice (Deuis and Vetter, 2016). Am. 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. As for the static hot plate test, cut off times should be carefully designed and strictly adhered to in order to avoid unnecessary nociceptive stimulation and tissue damage. Voluntary response bias is another disadvantage that comes with a small sample size. Eur. The amount of substrate displaced over a set period of time is recorded. Sci. 39, 16131623. This normally protective response, which is absent in these individuals, leads to frequent injuries and often results in higher mortality rates early in life (Bennett and Woods, 2014). Readership. Mechanical pressure is applied focally to the dorsal or plantar surface of the hind paw or tail, which is placed between a pointed probe tip and a flat surface. 86, 284288. Pain 59, 369376. Constraints on the tailflick assay: morphine analgesia and tolerance are dependent upon locus of tail stimulation. TRPM8 mechanism of cold allodynia after chronic nerve injury. Pain in rodents is associated with decreased burrowing behaviors. Burrowing behavior as an indicator of post-laparotomy pain in mice. Weight bearing as an objective measure of arthritic pain in the rat. Pharmacological interrogation of a rodent forced ambulation model: leveraging gait impairment as a measure of pain behavior pre-clinically. Despite some degree of uncertainty about the validity of the anthropomorphization of pain in animals, the capacity to experience pain and distress, particularly resulting from procedures or conditions that would cause pain and distress in humans, must be assumed unless there is evidence to the contrary. 96, 312317. doi: 10.1016/j.jneumeth.2015.08.010, Brenner, D. S., Golden, J. P., and Gereau, R. W. IV (2012). Nociceptive responses to high and low rates of noxious cutaneous heating are mediated by different nociceptors in the rat: behavioral evidence. 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). White, V.M.D., M.S., DACLAM, Dipl. The Hargreaves test is usually carried out using a glass bottom enclosure, which can be heated to minimize errors arising from heat sink effects. 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. 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. 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. (2000). Pain 16, 485495. 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. Sci. The 50% threshold is then calculated using the formula: 50% threshold (g) = 10(X+kd)/104, where X = the value (in log units) of the final von Frey filament, k = tabular value for the response pattern (see Appendix 1 in Chaplan et al., 1994) and d = the average increment (in log units) between von Frey filaments. Spared nerve injury: an animal model of persistent peripheral neuropathic pain. 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). 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. Acetylsalicylic acid, paracetamol and morphine inhibit behavioral responses to intrathecally administered substance P or capsaicin. Rotation of the handle of the hand-held device initiates heating from room temperature to 60C at a rate of 2.5C/s, which is terminated automatically by paw withdrawal, removal of the probe from the paw by the operator, or on reaching a predetermined cut-out (usually 60C; Figure 3D). Characterization of the foot withdrawal response to noxious radiant heat in the rat. Analysis of behaviors in unrestrained animals using automated technologies is increasingly being used to study non-stimulus evoked pain in rodents. 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). 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. doi: 10.1371/journal.pone.0039765, Brenner, D. S., Golden, J. P., Vogt, S. K., and Gereau, R. W. (2015). 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. Lancet Neurol. Assoc. Differentiating analgesic and non-analgesic drug activities on rat hot plate: effect of behavioral endpoint. (1988). Therefore, there is an urgent need to increase our understanding of the underlying mechanisms of pain and to develop new treatments. Comprehensive thermal preference phenotyping in mice using a novel automated circular gradient assay. Although animal models of pain have undoubtedly provided key advances, the advantages and disadvantages of each model and behavioral test should be taken into account to obtain objective and meaningful results that will improve our understanding and management of pain. 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). Assoc. Lancet 353, 19591964. Behav. 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). 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). doi: 10.1093/neuonc/nou048, Deuis, J. R., and Vetter, I. Detection of cold pain, cold allodynia and cold hyperalgesia in freely behaving rats. 119, 481488. While the plantar surface of the hind paw is the most commonly used area for testing, other areas of the body, including the dorsal surface of the hind paw or the abdomen can also be used (Minett et al., 2014). (A) Acetone evaporation test. 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. 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. 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). 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. However, it is unclear to what degree changes in gait in rodent models reflect altered pain or nociception, or conversely, anti-nociception or analgesia. Rats are an ideal choice for many labs due to their physiological similarity to humans. Activation of opioid receptors in cutaneous nerve endings by conorphin-1, a novel subtype-selective conopeptide, does not mediate peripheral analgesia. In cases of sensory loss, hypoalgesia may be present, which is defined as decreased sensitivity to a nociceptive stimulus. Eur. doi: 10.1016/j.neulet.2012.07.017. (2014). The time spent on the test plate relative to the reference plate is measured over a set period of time. (2015). One area where rats have contributed to major insights is the field of cerebral palsy research. (2013). Any long-term research involves mice and rats because of this unique aspect to the research. While forepaw withdrawal often occurs first, hind paw withdrawal or licking is considered to be a more reliable indicator of nociception, as the forepaws are frequently used in grooming and exploration and are not consistently in contact with the metal surface (Woolfe and Macdonald, 1944; Minett et al., 2011). When rodents are unwell, the amount of material removed from the burrow is decreased. The force at which this response occurs is recorded automatically by the apparatus and is designated as the paw withdrawal threshold. Computerized analysis of audible and ultrasonic vocalizations of rats as a standardized measure of pain-related behavior. Scientists often cite the similarities between animals and humans as the rationale for animal experimentation. 160, 15731576. Efficacy of drugs with different mechanisms of action in relieving spontaneous pain at rest and during movement in a rat model of osteoarthritis. 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. Ultrasound vocalisation by rodents does not correlate with behavioural measures of persistent pain. 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. Rodents are placed individually in a small cage with a barred floor. | All rights reserved. Neurosci. Sci. Am. Boehmerle, W., Huehnchen, P., Peruzzaro, S., Balkaya, M., and Endres, M. (2014). *Correspondence: Irina Vetter, i.vetter@uq.edu.au, View all Researchers and scientists conducting surveys and performing experiments must adhere to certain procedural guidelines and rules in order to insure accuracy by avoiding sampling errors such as large variability, bias or undercoverage. For example, in the spared nerve injury model, the tibial and common peroneal nerves are axotomized, leaving only the sural nerve intact. All we can possibly learn from high-dose experiments is what might happen in overdose situations. Pain 7:37. doi: 10.1186/1744-8069-7-37, Hunskaar, S., Fasmer, O. A modified Hargreaves method for assessing threshold temperatures for heat nociception. Many of these original stocks harbored a variety of natural, or indigenous, pathogens. 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). 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. (1974). Gait and weight bearing of rodents can be analyzed as a surrogate measure of nociception and are typically considered measures of non-evoked or stimulus-independent pain. Typically, the experimental setup consists of a test plate at a fixed temperature (usually between 5C and 55C) that is placed adjacent to a reference plate at neutral temperature (usually between 25C and 30C; Figure 5A). Ann Neurol 9(2): 131-41. The time taken to settle in a temperature zone and/or the temperature of the chosen zone is recorded. A soft transducer ensures minimal vibration and reduces touch-on responses. J. Pharmacol. . 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. Painful and painless channelopathies. Some of these parameters are altered in rodent models of pain, making gait analysis a method that is increasingly used to quantify non-stimulus evoked or spontaneous nociception in rodents. Figure 6.Methods used to assess non-stimulus evoked pain behaviors in rodents. Weight bearing as a measure of disease progression and efficacy of anti-inflammatory compounds in a model of monosodium iodoacetate-induced osteoarthritis. In nave C57BL/6 mice, the paw withdrawal temperature occurs at ~50C, and in unilateral models of inflammation the paw withdrawal temperature reduces to 4344C, providing a sufficient window to detect heat allodynia as well as hypoalgesia (Deuis and Vetter, 2016; Deuis et al., 2017a). As for heat pain, these values are likely influenced by a number of factors including ambient temperature, rate of cooling and anatomical location. engagement in scientific research and . doi: 10.1016/j.pain.2006.01.041, Roughan, J. V., Bertrand, H. G., and Isles, H. M. (2016). Life Sci. 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. doi: 10.1016/0165-0270(94)90144-9, Chapman, C. R., Casey, K. L., Dubner, R., Foley, K. M., Gracely, R. H., and Reading, A. E. (1985). (2005). In the case of researchers conducting surveys, for example, sample size is essential. Hyperalgesia and allodynia: peripheral mechanisms. The software records the duration of different behavior types, including movement, grooming and rearing behaviors. 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. Methods 219, 4151. Toxicol. Osteoarthr. doi: 10.1016/0024-3205(84)90575-7, Zimmermann, K., Deuis, J. R., Inserra, M. C., Collins, L. S., Namer, B., Cabot, P. J., et al. Heat thresholds in humans can be determined by applying a metal probe to the skin that increases in temperature (starting at 32C) until a warm-sensation threshold and heat-pain threshold is reached. 14:14. doi: 10.1186/1471-2474-14-14, Pertovaara, A., Kauppila, T., and Hmlinen, M. M. (1996). Weight distribution between the hind paws is recorded. (2007). An experimental model for peripheral neuropathy produced by segmental spinal nerve ligation in the rat. (D) Thermal probe test. A disadvantage of the Hargreaves test is that the paw withdrawal time is recorded rather than directly measuring the paw withdrawal temperature. 80, 300307. 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. A Guide to Research Rodent Housing William J. J. Musculoskelet. In recent years mice have been used with increasing frequency, to take advantage of genetically modified lines as well as the relatively lower costs compared with rats. 150, 165171. Appreciation that the human pain experience encompasses multiple stimulus modalities, distinct molecular mechanisms and sensory, motor, vegetative, emotional, motivational components should highlight the need for carefully designed experiments that take this complexity into consideration. Pharmacodyn. 52, 34703478. Life Sci. This raises a moral issue in animal experimentation. One way to evaluate nociception in non-communicating subjects is through observation of behavior. 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). Cybersex and pornography addiction. J. Neurosci. Therefore grimace scales have the potential to monitor and improve the welfare of animals used not only in research, but also in farming and industry. 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). ingenious was very competitively priced compared to other reputable transgenic companies. doi: 10.1016/s0165-0270(03)00241-3, Balayssac, D., Ling, B., Ferrier, J., Pereira, B., Eschalier, A., and Authier, N. (2014). 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.

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