Pain & Heat

The International Association for the Study of Pain (IASP) defines pain as “An unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage.” Over time many different approaches to defining pain have been seen throughout literature. However, the most important aspect of pain, in order to assess and treat it, is its classification.

In general, pain can be classified as

• Nociceptive Pain: This type of pain is normal and acts as an early-warning response to noxious insult or injury of tissues. This pain serves a protective purpose to prevent the body from injuring itself, and when engaged results in overruling of other neural responses.
• Inflammatory Pain: Similar to nociceptive pain, inflammatory pain is also adaptive and protective in nature. The pain results from the activation and sensitization of the nociceptive pain pathway to assist with the healing process of injured tissues or infection.
• Pathological Pain: Unlike the above two types of pain, pathological pain is maladaptive in nature. The pain is caused by abnormal functioning of the nervous system as a result of a primary lesion or disease in the somatosensory nervous system.

Further, depending on the duration of pain experienced, pain can be classified as acute and chronic pain. Acute pain tends to be a defensive response which usually lasts less than 3 to 6 months; however, chronic pain is characterized by longer durations (more than 3 to 6 months) and may be considered a diseased state. Improper treatments of pain can sometimes result in survived pain, and in some individuals, acute pain flare can superimpose on underlying chronic pain. Other classifications of pain are based on the location of the pain.

The multidimensional experience that pain evokes makes experimentation using humans challenging, in addition to being ethically limiting, due to the subjective nature of the experience. This makes animal models of pain a crucial part of the research. The different types of pains in an animal can be evoked using various approaches such as thermal, mechanical, chemical, or using disease models. Pain in animal models is usually assessed based on reflexive or non-reflexive measures. However, avoidance behavior-based assays such as Conditioned Place Avoidance, Place Escape-Avoidance Paradigm, and Thermal Escape Test can also provide useful insights into pain related-behaviors.

2.1 Thermal Pain Assays

Thermal pain assays are commonly used to assess pain responses to noxious heat or innocuous cold temperatures. These pain assays usually involve direct application of the thermal stimuli to the plantar surface. Reflexive behaviors such as paw licking, jumping, and withdrawal of paw or tail are used to assess the pain thresholds of the animal. However, the observed behaviors may not always be resultant of hypersensitivity to the painful stimuli.

Tail Flick Test

The earliest mention of the Tail Flick test can be traced to D’Amour and Smith’s 1941 experiment of loss of pain sensation in rats. The test involves the application of the heat stimulus to the tail of the rodent. The application of the stimulus can be achieved either by dipping the tail into water (maintained at a predetermined temperature) or by using radiant heat stimulus to a part of the tail. This test requires the subjects to be habituated to handling and thus, the results in the test can be affected by the subject’s ability to acclimatize to restraint. (For restrainers click here)

Hot Plate

The Hot Plate was described by Woolfe and Macdonald in 1944 in their paper investigating the analgesic effect of pethidine hydrochloride. The apparatus involves a hot plate that can be maintained at a constant temperature or varying temperatures as required by the experiment. The animal in the apparatus is confined to the hot plate arena using a clear acrylic cylinder.

Hot/Cold Plate

The Hot/Cold Plate combines a hot plate and a cold plate into one unit. This combination allows of assessment of nocifensive behaviors resulting from the application of either noxious hot temperatures or innocuous cold temperatures.

Plantar Test Hargreaves’s Apparatus

Hargreaves and colleagues first described the Hargreaves Plantar test in 1988 (Hargreaves et al., 1988) in their assessment of cutaneous hyperalgesia resulting from thermal stimulation. The apparatus consists of glass enclosures with controllable emitter/detector vessel to manipulate the heat intensity. Unlike other thermal pain assays that apply heat stimulus to the entire plantar surface, the Hargreaves apparatus uses a directed heat stimulus at a particular point on the plantar surface.

Thermal Gradient

The Thermal Gradient apparatus is composed of metal plates placed adjacent to each individual plate permitting the creation of different temperature zones. The principle behind the design of the apparatus is that the subjects will show a preference for a temperature zone based on their thermal sensitivity, which is influenced by their nociceptive states. The Thermal Gradient, similar to the Hot/Cold plate allows assessment of pain behaviors and responses to both cold and hot temperature stimuli; however, simultaneously.

2.2 Mechanical Pain Assays

Mechanical pain assays are commonly used in the assessment of mechanical allodynia and hyperalgesia. One of the earliest methods of mechanically-induced pain was through the manual application of a filament tip with gradually increasing force. This method was adopted from Max von Frey’s research using human patients that were assessed for their pressure sensitivity using calibrated horse hair (von Frey, 1896). Observed behaviors to mechanical stimulation include withdrawal, licking, or shaking of the paw. However, it is unclear whether the responses are, in fact, responses to mechanical pain or mechanical sensibility.

Electric von Frey

The Electric von Frey is a modern take on the von Frey monofilaments designed by Maximilian von Frey as an esthesiometer in 1896 (Pearce, 2006). The filaments are applied perpendicularly to the skin of the plantar surface in an up-down method until they buckle. The Electric von Frey uses a single filament that permits a range of pressure application. Unlike other tests, the von Frey test does not require handling or restraining of the animal, which reduces the influence of stress on the animal.

Randall-Selitto Test

Similar to the von Frey filament, the Randall-Selitto test uses a pressure applicator to quantify mechanical pain sensitivity. The method was developed by Randall and Selitto in 1957 to evaluate the effect of analgesics on inflamed tissues. Usually, the test involves the application of pressure on the paws (hence the name Paw-Pressure Test), but it can also be used to apply pressure on the tails of the rodents. The test provides a sensitive and rapid measure of pain sensitivity. This test requires the subjects to be habituated to restraining. (For restrainers click here)

2.3 Chemical Pain Assay

Chemical pain assays involve the application or injection of noxious chemical stimuli to induce pain. Depending on the approach observed behaviors could include flinching, licking, and biting of the affected paw.

Formalin Test

The Formalin Test was developed by Dubuisson and Dennis (1977) to study the analgesic effects of morphine, meperidine, and brain stem stimulation. The test involves the subcutaneous injection of dilute formalin into the paw of the animal and observing the pain-related behavioral responses. The test produces a response in two discrete stages, the acute phase, and the tonic phase, within a limited duration of approximately 1 hour.

Acetone Evaporation Test

The Acetone Evaporation Test was first described in 1994 (Carlton, Lekan, Kim, & Chung, 1994) and is used in the assessment of cold allodynia. The test involves dabbing or spraying the plantar surface of the paw of the subject with acetone and observing the pain-related behaviors as the chemical evaporates.

2.4 Operant Pain Assays

Operant pain assays are sensitive, have better validity, and encompass psychological and affective dimensions of human pain. Unlike traditional reflex-based approaches,  operant pain assays involve the observation of voluntary behavior. Outcome measures in these assays usually include the duration of contact with the painful stimuli and latency of withdrawal.

OroFacial Pain Assessment Device

The Orofacial Pain Assessment Device (OPAD) was developed by Neubert and colleagues (2005) as an operant system of pain assessment that relies on voluntary behavior. The device design utilizes the reward-conflict paradigm wherein the subject makes a conscious decision of its pain threshold in order to gain the reward. The apparatus is composed of Peltier-based thermode that allow variation of applied temperature in order to assess thermal pain. Additionally, metal wires allow assessment of mechanical pain sensitivity, which can also be simultaneously measured along with thermal pain.

Thermal Escape Test

The Thermal Escape Test uses a shuttle-box paradigm that allows the subject to explore freely. The apparatus includes two chambers that can be individually set to either hot or cold nociceptive temperature or other temperature combinations. The apparatus was developed by Mauderli, Acosta-Rua, and Vierck, 2000 to overcome the short-coming of electrical stimulation-based shuttle-box tests (changing grid floor polarities and current densities due to animal movement).

Conditioned Place Avoidance

The Conditioned Place Avoidance paradigm uses the same principle as the Conditioned Place Preference. The apparatus usually consists of two or three compartments of which one serves as the neutral compartment. Animals are preconditioned to the conditioning chambers following an injection of a noxious substance that induces pain. The following day the subjects are reintroduced into the apparatus without the injection. This test allows observation of avoidance behavior that is associated with pain. However, the test requires significant learning, which can interfere with pain-related behavior observation. Further, learning can be impaired due to pain or analgesic treatment.

Place Escape-Avoidance Paradigm

Similar to the Conditioned Place Avoidance paradigm, the Place Escape-Avoidance Paradigm uses a two-chamber apparatus to allow observation of pain-related behavior. The paradigm involves stimulating, using noxious stimulus, the painful paw in one of the chambers and the non-painful paw in the other chamber. Subjects can be seen opting for the chamber where the non-painful paw is stimulated to escape the unpleasantness of the other chamber.

Apart from eliciting neural responses, pain also manifests itself in deterioration of emotional health and other comorbidities which may not be obvious at first. Pain-stimulated behavior, pain-depressed behaviors, and functional disability measures enhance the understanding of pain and how it affects the quality of life. Monitoring of cage behaviors using a tracking and recording system such as the Noldus EthoVision XT can also provide many insights into the day-to-day functioning of the animal models of pain

3.1 Pain-Stimulated Behaviors

In animal models, pain may be expressed via vocalization or changes in facial features. Grimace scales are useful for scoring the subjective intensity of pain. In rodent models, the Mouse Grimace Scale (Langford et al., 2010) and the Rat Grimace Scale (Sotocina et al., 2011) are commonly used. Another common behavior is the Ultrasonic Vocalizations, which has been observed in rodents in response to acute pain (Kurejova et al., 2010; Williams, Riskin, & Mott, 2008).

3.2 Pain-Depressed Behaviors

Pain-depressed behaviors draw clinical parallelism to human behaviors. These behaviors involve the fluctuation in the frequency of daily activities (apart from drinking and eating) as a result of pain. In humans, these behaviors are usually assessed using self-assessment questionnaires (For digital health research tools visit Qolty), while in animals it is done via observation of behaviors such as burrowing (see Burrowing Tube Test). While depressive behaviors may be caused by factors other than pain, they still provide valuable data when assessing the effect of pain on quality of life. Behavioral observations can be done by observing home cage behaviors using special detector plates and/or camera observations (see also Visual Burrow System). Apart from home cage behaviors, other assays such as within-cage Wheel Running (see Activity Cage), nest-building behavior, sleep pattern, and Sucrose Preference test can also provide helpful insights.

3.3 Functional Disability

Pain can result in decreased physical function resulting in pain disability. In animal models, the popular measure for assessing functional disability is weight-bearing, which can be analyzed using Gait Test and fTIR Walkway. Other assays include Grip Strength, and observation of locomotion (see Open-Field Test).

Animals play a crucial role in expanding the understanding of pain, biological mechanisms, and pain-related behaviors and morbidities. While it is impossible to conduct pain research without causing any stress or harm to the animal, efforts should be made to perform all investigations as ethically as possible. Zimmermann (1983) outlined the following guidelines for pain experiment involving conscious animals,

• Experiments should be reviewed by scientists and lay-person before execution.
• When possible, in the case of non-invasive stimuli-based procedures, investigators should try the stimulus on themselves.
• Physiological and behavioral parameters should be measured to assess the deviation from normal behavior in animals. These measures should be included in the manuscript.
• Measures to ensure that the animal is exposed to only the minimal necessary pain in acute or chronic pain studies should be taken.
• As long as the experimental aim is not affected, animals should be allowed treatment for pain relief or self-administer analgesic in chronic pain studies.
• In studies involving the use of a neuromuscular blocking agent, it must be ensured that the animal is either treated with a general anesthetic or an appropriate procedure to eliminate sensory awareness.
• The number of animals used and the duration of the experiments should be kept to a minimum.

Apart from the above guidelines, efforts should be made to ensure the overall wellbeing of the animals in the laboratory. Animals should not be subjected to unnecessary stress or mishandling at any time.

Animal models allow the modeling of different pains and related disorders and diseases. Though they do not serve as an exact match for studying human pain, they are an ideal and feasible approach to expanding the understanding of pain and development of treatments.

Experimental designs of pain study vary depending on the etiology, area of pain induction, and duration; regardless, most assays measure either reflexive or non-reflexive responses. Apart from pain-induced responses which provide an understanding of biological processes involved, other pain-related behaviors can also provide further insights. Since pain is a multidimensional experience that is both sensory and emotional, expanding research into areas investigating the effect of pain on physical activity and emotional health allow for the development of better treatments.

Though animals must be subjected to painful experiences, efforts must be put to ensure that all procedures are ethical. Care must be taken to not subject the animals to any unnecessary harm, stress, or inconvenience. Further, where possible, the number of animals used should be reduced, and the pain intensity should not exceed the appropriate minimum required.

1. Carlton, S. M., Lekan, H. A., Kim, S. H., & Chung, J. M. (1994). Behavioral manifestations of an experimental model for peripheral neuropathy produced by spinal nerve ligation in the primate. Pain, 56(2):155-66.
2. D’Amour F. E., & Smith D. L. (1941). A method for determining loss of pain sensation. Journal of Pharmacology and Experimental Therapeutics, 72, 74–79.
3. Dubuisson, D., & Dennis, S. G. (1977). The formalin test: a quantitative study of the analgesic effects of morphine, meperidine, and brain stem stimulation in rats and cats.Pain 4(2): 161-174.
4. Gregory, N. S., Harris, A. L., Robinson, C. R., Dougherty, P. M., Fuchs, P. N., & Sluka, K. A. (2013). An Overview of Animal Models of Pain: Disease Models and Outcome Measures. The Journal of Pain, 14(11), 1255–1269. Doi:10.1016/j.jpain.2013.06.008
5. Hargreaves, K., Dubner, R., Brown, F., Flores, C., & Joris, J (1988). A new and sensitive method for measuring thermal nociception in cutaneous hyperalgesia. Pain, 32(1):77-88.
6. Kumar, K. H., & Elavarasi, P. (2016). Definition of pain and classification of pain disorders. Journal of Advanced Clinical & Research Insights,3, 87-90. doi:10.15713/ins.jcri.112
7. Kurejova, M., Nattenmüller, U., Hildebrandt, U., Selvaraj, D., Stösser, S., & Kuner, R. (2010). An Improved Behavioural Assay Demonstrates That Ultrasound Vocalizations Constitute a Reliable Indicator of Chronic Cancer Pain and Neuropathic Pain. Molecular Pain, 6, 1744–8069–6–18. doi:10.1186/1744-8069-6-18
8. Langford, D. J., Bailey, A. L., Chanda, M. L., Clarke, S. E., Drummond, T. E., Echols, S., … Mogil, J. S. (2010). Coding of facial expressions of pain in the laboratory mouse. Nature Methods, 7(6), 447–449. doi:10.1038/nmeth.1455
9. Mauderli, A. P., Acosta-Rua, A., & Vierck, C. J. (2000). An operant assay of thermal pain in conscious, unrestrained rats. Journal of Neuroscience Methods, 97(1), 19–29. doi:10.1016/s0165-0270(00)00160-6
10. Mogil, J. S. (2009). Animal models of pain: progress and challenges. Nature Reviews Neuroscience, 10(4), 283–294. doi:10.1038/nrn2606
11. Mogil, J. S., Davis, K. D., & Derbyshire, S. W. (2010). The necessity of animal models in pain research. Pain, 151(1), 12–17. doi:10.1016/j.pain.2010.07.015
12. Neubert, J. K., Rossi, H. L., Malphurs, W., Vierck, C. J. Jr, & Caudle, R. M. (2006). Differentiation between capsaicin-induced allodynia and hyperalgesia using a thermal operant assay.Behavioral Brain Research, 170(2):308-15.
13. Pearce, J. M. S. (2006). Von Frey’s pain spots. Journal of Neurology, Neurosurgery & Psychiatry, 77(12), 1317–1317. doi:10.1136/jnnp.2006.098970
14. Randall, L.O., & Selitto, J.J. (1957). A method for measurement of analgesic activity on inflamed tissue. Archives Internationales de Pharmacodynamie et de Therapie, 111, 409–419.
15. Sotocina, S. G., Sorge, R. E., Zaloum, A., Tuttle, A. H., Martin, L. J., Wieskopf, J. S., … Mogil, J. S. (2011). The Rat Grimace Scale: A Partially Automated Method for Quantifying Pain in the Laboratory Rat via Facial Expressions. Molecular Pain, 7, 1744–8069–7–55. doi:10.1186/1744-8069-7-55
16. Swieboda, P., Filip, R., Prystupa, A., & Drozd, M. (2013). Assessment of pain: types, mechanism and treatment. Annals of Agricultural and Environmental Medicine, Spec no. 1:2-7.
17. Tappe-Theodor, A., & Kuner, R. (2014). Studying ongoing and spontaneous pain in rodents – challenges and opportunities. European Journal of Neuroscience, 39(11), 1881–1890. doi:10.1111/ejn.12643
18. von Frey, M. (1896). On the Use of Stimulus Hairs. Journal of Neuroscience Methods, 71–131.
19. Williams, W. O., Riskin, D. K., & Mott, A. K. (2008). Ultrasonic sound as an indicator of acute pain in laboratory mice. Journal of American Association for Laboratory Animal Sciences, 47(1):8-10.
20. Woolf, C. J. (2010). What is this thing called pain? Journal of Clinical Investigation, 120(11), 3742–3744. doi:10.1172/jci45178
21. Woolfe, G., & Macdonald A. D. (1944). The evaluation of the analgesic action of pethidine hydrocholoride (Demerol). Journal of Pharmacology and Experimental Therapeutics, 80, 300–307
22. Zimmermann, M. (1983). Ethical guidelines for investigations of experimental pain in conscious animals. Pain, 16(2), 109–110. doi:10.1016/0304-3959(83)90201-4