Animal Models of Pain - Clinical Tree

SUMMARY

Animal models of pain, usually rodents, support basic research on physiological and pathophysiological mechanisms, as well as applied research in drug discovery. Quantification of pain and analgesia relies on responses evoked by physical or chemical stimuli and modification of motor behavior in normal animals or following a variety of forms of injury. Electric stimuli are easy to regulate but bypass the nociceptor endings, recruit non-nociceptor afferents more easily than nociceptive ones, and have unpredictable effects on tissue. Heat stimuli can be tailored to activate different populations of nociceptors selectively but, depending on the heat source, may be difficult to control. Heat sensitivity is commonly tested as the latency to withdrawal from a radiant heat beam. Cold may be applied by a drop of acetone, by ethyl chloride spray, or by a cold plate, the latter delivering a mixed cold and mechanical stimulus. Tactile sensitivity may be tested with von Frey filaments, mechanical hyperalgesia by pinprick, and deep pressure by devices generating progressive force. Mechanical stimuli are generally difficult to control. Chemical stimuli activate specific transduction mechanisms and/or produce persistent activation of nociceptors, as in the formalin test. Popular models of chronic pain typically measure hypersensitivity caused by monarthritis of the ankle or knee joint or traumatic neuropathy of the sciatic nerve, but there are many others that exhibit more or less face validity of clinically relevant pain states. Minor differences in protocols may have a great impact on readouts from the models, and thus the need for improving experimental and reporting standards has been recognized. Current challenges in animal modeling include quantification of ongoing pain and implementation of pharmacokinetic parameters in pharmacological experiments.

Introduction

Animal modeling of the complex sensory, emotional, and ultimately subjective experience of pain poses a number of challenges. First, there is limited understanding of the pathophysiological mechanisms underlying the heterogeneous conditions that are the aim of the modeling. Other fundamental challenges are related to understanding the physical characteristics and physiological consequences of the stimulus used in the modeling paradigm, interpreting behavior as an expression of pain (or some other sensory experience), and understanding the relationship between stimuli and responses. Under physiological conditions a painful stimulus is likely to be transient, such as a pinprick or hot object, but under pathological conditions, particularly if chronic, pain may be generated or amplified from processes within the affected organs, be it an inflamed joint or an injured and dysfunctional nervous system. The processing of noxious signals and the experience of pain are certainly modulated by activity in the central nervous system. External stimuli are useful in probing the internal state, but we cannot a priori assume a direct relationship between evoked responses and the pain experience. Our methodology to quantify ongoing pain in animals is much less advanced than the armamentarium available for studying evoked responses, but there is growing interest in bridging the gap, and some approaches will be discussed in following sections.

The experience of pain can be inferred from an animal’s behavior. This is, of course, also the case with human beings not able to verbalize or communicate in the observer’s language. Although we may intuitively find it easier to interpret behavior displayed by a member of our own species, in reality, both verbal and non-verbal communication between humans is inherently imprecise and subject to interpretation ( ). People as well as animals may have reasons to exaggerate or suppress their expression of pain. Consequently, strictly objective measures of pain may not be achievable whether in animals or in human patients, and interpretation of behavior and verbal report will continue to play an important role in understanding and measuring pain. Some components of the pain experience may be unique to humans and therefore not possible to model in animals. This is particularly the case with chronic pain, where, for example, cognitive and emotional adaptation to the pain condition is less likely to be similar between species than, for instance, plastic changes in descending inhibition driven by noxious input. Recognizing these limitations is a prerequisite for effective use of animal models.

It is nevertheless unlikely that the ability to feel and experience pain, which has obvious survival value, would differ fundamentally between mammals. This is a basic assumption for animal modeling and has ethical implications. There is usually no conflict between good scientific practice and ethical treatment of laboratory animals, but it is important to formulate a clear, testable hypothesis, use adequate experimental designs, and minimize suffering both in terms of the individual animal and with regard to the number of animals. Another aspect is that the results should be published or lead to scientific progress in some other way. Reluctance to publish negative findings may contribute to an overall bias in the literature ( ) and possibly lead to unnecessary experiments.

Modeling may serve several purposes. Understanding the pathophysiology of pain with the aim of developing new prevention and treatment strategies, including novel analgesics, is probably the most cited motivation. Although the utility of animal modeling in analgesic drug development has been questioned ( ), current models used carefully and with due attention to supporting data from the use of other methods have provided invaluable input to this process and will continue to do so in the foreseeable future ( , ). Animal models also expand our understanding of the normal physiology of nociceptive signaling—from molecular mechanisms to integrated systems. Basic science is valuable in its own right and an important foundation for applied research and generation of hypotheses with practical implications.

The number of animal models available for research on pain and analgesia is large and growing. Any model represents a simplification of the modeled condition, and it is important to recognize that the utility and validity of a model are fundamentally related to the specific hypothesis under investigation. Although a single model should not be expected to represent all aspects of a complex condition such as chronic neuropathic pain in humans, a set of models and supporting methods may provide decisive information about critical pathophysiological mechanisms.

Pain modeling is usually thought of in terms of behavior, but electrophysiology, functional imaging, biochemical biomarker analysis, genetics, and analysis of exposure and pharmacokinetics provide information that is indispensable for the elucidation of pain processes. A combination of several approaches is the most powerful way to address this complex area. This chapter focuses on commonly used behavioral methods, but other methodological tools will be mentioned or referred to as needed for discussion. Modeling will be described in terms of the stimuli used to evoke quantifiable responses and behavior, with critical discussion of some of the more popular methods of stimulation and quantification, and finally the manipulation that creates the relevant pathophysiology (the model per se). Rodent models are the main focus of this review, but it is appreciated that species differences in physiology, anatomy, and pathophysiology mean that other species are needed, such as for analysis of primary afferent functions translatable to human microneurography studies ( ) or to overcome differences in receptor pharmacology ( ).

Common Stimuli and Quantification of Evoked Responses

Nociceptive tests based on withdrawal responses evoked by stimuli at or near threshold intensity (e.g., thermal assays such as the tail-flick and hot plate tests) are frequently referred to as models of acute pain. This can be confusing since acute pain in clinical terminology typically refers to pain caused by accidental injury or surgery and involves inflammatory and sensitizing mechanisms not present in the basic implementations of these tests. Similarly, animal models of chronic pain are usually carried out in a time span of minutes to a few weeks, whereas in the clinic, chronic pain refers to conditions lasting at least 3 months. This distinction is not just semantic since some pathophysiological processes require more than a few weeks to develop. The formalin test may mimic some features of a particular chronic pain condition (e.g., paroxysmal peripheral input) but will not emulate, for example, long-term central nervous system plasticity. Conversely, inflammatory processes inherent in the model or its initiation may be more typical of a postoperative stage than of chronic neuropathy, for instance.

Quantification of pain and analgesic effect in animal models usually relies on responses evoked by physical or chemical stimuli or modification of motor behavior observed after tissue injury or inflammation. The use of controlled stimuli such as heat, cold, electric shock, or injection of chemical mediators may facilitate the specific activation of discrete groups of primary afferents, which are classified as mechanonociceptors, polymodal nociceptors, and mechanically insensitive nociceptors and further subdivided according to transduction and conduction properties ( , , , , , ; see also Chapter 1 ). These primary afferents are complemented by others responding to non-noxious levels of intensity. For mechanistic studies, specific activation of certain fiber types is a great asset, but using highly standardized and discrete methods may result in data and conclusions valid only for a limited set of circumstances. The difference between painful and non-painful is often a matter of stimulus intensity, and the criteria for whether a response reflects pain or some other modality may be arbitrary, particularly when graded stimuli are used.

Electric Stimuli

Electric stimuli are currently rarely used in pain research except for electrophysiological experiments and conditioning paradigms but have in the past been applied extensively in paradigms such as the flinch-jump test, vocalization tests, and jaw-opening models ( ). Electric stimuli are quantifiable and time-locked and can be repeated without damaging the stimulated tissue. Although delivery of an electric stimulus is easy to control, the impact on the target tissue is affected by a number of factors ranging from differences in tissue impedance (which may be affected by behavioral strategies of the subject) to contamination of the grid through which the stimulus is delivered. These factors may be difficult to recognize and control for, particularly when using conscious, unrestrained, or lightly restrained animals. Electric stimuli bypass the peripheral receptors and recruit all types of cutaneous nerve fibers, depending on stimulus intensity, with large-diameter fibers being excited at lower intensities than fine-diameter nociceptors. This means that a mixture of sensory modalities will be activated with a given stimulus, possibly engaging central inhibitory functions in addition to nociception. Human subjects tend to find electric stimuli aversive regardless of whether they are painful. In rodents, stimuli just above the detection threshold elicit an orienting response, and higher intensities elicit progressively more forceful responses. A pain threshold can be defined by using arbitrary criteria such as vocalization or jumping synchronized with the shock, but because of the aversive nature of electrical stimulation, even at low intensities, avoidance or escape responses are not sufficient to assume pain experience.

Heat

Heat is an adequate pain stimulus and a versatile modality that is used extensively in both animals and humans. The prototypical animal method based on radiant heat is the tail-flick test originally described by , which was preceded by the extensive use of radiant heat stimuli in human psychophysical studies ( ). Various methods of heating are listed in Table 11-1 .

Table 11-1

Commonly Used Heat Stimuli

STIMULUS	TEMPERATURE INCREASE	FACTORS AFFECTING STIMULUS INTENSITY AT RECEPTORS	APPLICATION
Radiant	Proportional to the square root of time	Initial skin temperature Skin pigmentation	Tail and paw withdrawal tests
Thermode	Proportional to time	Contact with skin	Mostly human models
Hot water	Nearly instantaneous at the surface, gradual in deeper layers	Initial skin temperature	Tail- and paw-dip tests
Constant-temperature hot plate	Rapid at the surface, gradual in deeper layers	Initial skin temperature Motor activity Contamination of the floor	Hot plate test
Ramped hot plate	Proportional to time	Contamination of the floor	Hot plate test
Laser	Nearly instantaneous	Skin thickness	Mostly human models

The rate of increase in temperature in the exposed tissue depends on the type of heat source and the energy supplied. Most methods used in animal research are significantly influenced by the initial temperature of the skin. Depending on the tissue temperature achieved and the rate of heating, different classes of nociceptors are recruited, which can be used purposefully for mechanistic studies or contribute to unwanted variability and false interpretations if not adequately considered ( ). C fibers are activated at lower temperatures than Aδ fibers are, and slow heating rates will primarily recruit C fibers whereas more rapid heating will favor Aδ nociceptors ( , , ).

Heat stimuli are usually delivered by means of radiation. With this stimulus modality, transfer of heat to the layers of the nociceptive nerve endings is affected by a number of factors that need to be controlled. Skin color is important for heat absorption, and the heating rate can be accelerated by blackening of the skin ( ). Conversely, differences in skin color may be a source of variation and even a confounding factor if there are systematic differences between subjects ( ). As illustrated in Figure 11-1 , skin temperature at the outset of stimulation is another important factor when radiant heat is used ( , , ).

Figure 11-1, Relationship between initial skin temperature and latency to withdrawal when a radiant heat stimulus is applied.

Since its introduction, the radiant heat paw withdrawal method ( ) has rapidly become the standard method for measuring heat sensitivity in rodents, largely because it can easily be applied to the paw inflammation and sciatic neuropathy models of growing popularity. In this assay, a beam is projected through a transparent floor and (as in the tail-flick assay) the behavioral end point is the response latency, in this case withdrawal of the stimulated paw. Animals can be tested with a minimum of potentially stressful restraint, and the stimulus is directed to a specific and restricted part of the body while avoiding heterosegmental stimulation of nociceptors that might be a confounding factor, as in the hot plate test. The assay is, however, sensitive to certain factors, including posture, exact focus of the beam, initial temperature of the skin (which may be altered by inflammation or neuropathy, as well as by handling), conditions in the testing environment, and confounding pharmacological effects ( , , , ). Preheating the floor to a holding temperature above the normal range of skin temperature will reduce or eliminate the influence of initial skin temperature and allow better reproducibility of the stimulus function. Importantly, starting from a higher temperature makes it possible to apply a slower heating rate without causing impractically long response latencies, and these factors may tune the assay toward C fiber–mediated responses. Description of the method used in publications is frequently unclear regarding whether adequate controls have been established for these potentially important variables.

Heat stimuli can also be delivered by direct contact with a heat source. Thermodes are common in human experimental work and on anesthetized animals but for practical reasons are used less frequently in behavioral studies. Thermodes can be used at a predetermined fixed temperature or to deliver a linear increase in temperature from a defined holding temperature. In principle, this is also the case with the rising-temperature hot plate assay, in which the temperature is increased at a slow rate (typically 2–3°C/min from a temperature of 32°C), thereby allowing estimation of an approximate response threshold as the temperature at which a predetermined response (e.g., licking of a hindpaw) occurs ( ). However, because of the temperature gradient between the surface and the core, the observed threshold will be higher than the actual temperature at the nociceptors. The slow increase in temperature and the limited maximum temperature mean that the assay is likely to involve C fibers to a greater extent than conventional methods using a constant temperature source, as in the traditional hot plate assay or tail-dip and paw-dip assays. When compared with the conventional constant-temperature hot plate, it is also less sensitive to motor activity and stress ( ), but exposure of several body parts to the hot plate means that heterosegmental interaction can take place.

These acute thermal assays can be used to study physiological nociception, and their utility and limitations have been discussed more extensively elsewhere ( ). Since the recruitment of different classes of heat nociceptors and the relative degree of non-nociceptive thermosensor activation depend on the heating profile, minor differences in protocol may be physiologically and pharmacologically relevant. Traditional implementations of acute heat pain assays in rodents tend to favor responses mediated through Aδ-fiber stimulation.

It has long been recognized that methods using acute thermal stimuli are sensitive to agonists at the μ-opioid receptor but not to other types of opiates ( ). The hot plate assay is, for instance, capable of predicting the potency ranking of spinal opioid analgesics corresponding to clinical dosing for postoperative pain ( ). The actual potency estimates of morphine, for instance, may differ 20–50-fold in a single paradigm, depending on the heating profile ( ). The assays are not sensitive to non-opiate analgesics unless very high, perhaps toxic doses are administered ( , ).

Cold

Cold stimuli are used primarily in the context of neuropathic pain models and in work aimed at characterizing physiological and molecular mechanisms of cold perception and cold pain. Research on cold transduction has made great progress in the last 10–15 years, but the way that signaling in different afferent fiber types relates to tissue temperature and sensory quality is less clear than that for heat perception. Although understanding of how these factors contribute to pathological pain in chronic conditions is incomplete, it is clear that as for heat, both the rate of change in temperature and the actual tissue temperature determine the recruitment of different fiber types and thus the mechanisms activated by a cold stimulus ( , ).

The most common way of inducing cooling and pain in rodent behavioral models is probably the application of acetone. The test is also used clinically; the stimulus is normally perceived as innocuous but may evoke allodynia in a minority of chronic pain patients ( ). A typical testing protocol in the rat involves the application of a fixed volume of acetone to the plantar skin by means of plastic tubing and counting the frequency of responses, defined as brief withdrawal of the paw, obtained after a predefined number of evenly spaced applications ( ). Acetone spray and ethyl chloride spray are also used, thus adding a mechanical component to the stimulus, and the outcome variable is then usually the accumulated duration of paw withdrawal recorded for a period of up to 60 seconds after application ( , ). The degree of cooling and the contribution of the mechanical component may vary significantly between implementations of these methods, and therefore achieving reproducibility between different operators may be a challenge. Less frequently, cold sensitivity has been measured by means of a cold plate ( ) or cold water bath ( ), analogous to the heat stimulus paradigms, with latency to withdrawal or the frequency or duration of paw lifts being outcome measures.

Mechanical Stimulation

A diverse set of devices for mechanical stimulation exist, and we will discuss the most commonly used of these, von Frey filaments, which are used to deliver punctuate pressure. We also briefly discuss the pinprick method for punctuate hyperalgesia and approaches to measure deep pressure. In general, mechanical stimuli are difficult to apply in a standardized fashion even when standardized equipment is used, and the great number of devices and protocols make it difficult to compare results reported in different publications. In interpreting the data it is therefore important to understand the physiological consequences of the stimulation procedure in each case.

Von Frey Filaments and Similar Methods

Von Frey filaments were introduced in the 19th century to determine sensory thresholds in humans. The method is based on the principle that a monofilament will bend at a distinct force when applied perpendicular to a surface. A range of calibrated filaments can be used to calculate a response threshold or, alternatively, the response frequency ( ). When used in rodents, the animals are usually placed on a covered grid and probed from below, but in some cases the animals are restrained. Response is defined as brisk withdrawal of the paw, but a “hyperalgesia-like” response in which the animal elevates the paw for a second or more or shakes, grooms, licks, or chews the paw has been suggested to differentiate between aversive and non-aversive sensations when applied to neuropathic rats ( ). This exaggerated behavior is rarely seen in control animals unless a very thin probe is used.

Von Frey filaments are used extensively but are associated with a number of drawbacks. has provided a detailed analysis that can be summarized as follows:

The actual pressure on the skin will change during the application of a particular filament because contact surface area and geometry depend on the degree of bending and compliance of the tissue.
Force generation may differ from the nominal value even under standardized conditions and depends on application speed (slow application leads to slower buildup of force). Unpredictable off-axis forces are created as the fiber bends.
Inconsistency of use with no universally accepted procedure makes comparison of results difficult.
Bias may result from the obvious difficulty in blinding the experimenter to the filament used and, in some cases, to pretreatment of the animals.
Application of standard filaments to normal skin does not produce pain in humans (it is common practice in animal work to assign a cutoff value as baseline), and thin fibers may produce an itch.

These factors and the use of discrete filaments of fixed nominal bending force limit accurate estimation of thresholds and comparison of data between laboratories. further refers to anecdotal data suggesting a discrepancy between the effect of filament stimulation and a set of other, perhaps more relevant mechanical stimuli, such as light or hard stroking and vigorous rubbing of the affected paw.

Some of these problems can be minimized by proper technique, such as stopping stimulation immediately when the filament bends, but there are additional complications related to the mechanical instability of conventional filaments. They are usually made of hygroscopic material and their bending properties will change rapidly in response to normally occurring fluctuations in relative humidity; a No. 3 hair at 26% relative humidity may bend at higher force than a No. 6 filament at 56%. They are also affected by temperature and deformation. As mentioned earlier, contact area geometry plays a role in stimulus function, and other factors being equal, probes of smaller diameter will be more effective (see for references).

Using filaments made of non-hygroscopic material and with a fixed tip size can circumvent several of these problems ( , ). Force transducers based on von Frey–like devices (“electronic von Frey”; Fig. 11-2 ) are also available and facilitate standardization of stimulus application and avoid the problem of variable geometry and mechanical instability of the probe ( ). Standardized protocols that would reduce inter- and intraobserver variability have been suggested (e.g., , ), with some salient factors to consider being the duration of application, stimulus interval, degree of bending, and site of stimulation.

Figure 11-2, Response to mechanical stimulation in the spared nerve injury model.

Pinprick

Mechanical hyperalgesia is frequently tested by the pinprick method, which in its simplest form is performed by applying a sharp object such as a safety pin or an injection needle to the skin to cause an indentation but no penetration. The animals may be restrained for the procedure or, more commonly, placed on a grid. The response, either brisk withdrawal or a “hyperalgesia-like” response with sustained elevation, such as licking and grooming of the paw, can be quantified as the duration of such behavior in a predefined time frame ( , ). In human experimental work, a more standardized procedure has been devised in which weighted pinprick stimulators with a fixed flat contact area of 0.2 mm in diameter produce force between 8 and 512 mN, which can be used to calculate threshold and stimulus response functions ( ).

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