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36 Animal Models of Nociception and Pain

JAMES D. ROSE AND C. JEFFREY WOODBURY

ABSTRACTResearch on the neurobiological bases of nociception and pain

and related investigations of potential therapies require great reli-ance on animal models. There are unique challenges in the devel-opment of well-validated models in this field because of the distinction between nociception, the processing and response to potentially pain producing stimuli by lower levels of the nervous system, and pain, the conscious result of nociceptive stimulus processing by the cerebral cortex. The most frequently used models actually represent tests of nociception only and are appro-priate for investigating diverse pathophysiological processes that cause nociceptive activity in peripheral tissues, nerves, the spinal cord, or subcortical regions. However, because human pain is a complex end result of nociception and consciousness-dependent processes, models intended to address pain must be validated for this purpose. Models assessing processes related to pain are rela-tively rare and more difficult to validate and use than those rele-vant only for nociception. A failure to recognize the pain–nociception distinction has significant practical consequences for successful extrapolation of results from laboratory to clinical practice.

Key Words: Nociception–pain dichotomy, Construct valid-ity, Neocortex, Nocifensive behavior, Neural substrate.

THE NEED FOR ANIMAL MODELS OF NOCICEPTION AND PAIN

Pain research with human subjects is productive on many fronts, as shown by the large and diverse literature surveyed in the most recent edition of Wall and Melzack’s Textbook of Pain.1

A particularly prominent area of progress is in the use of brain imaging methods such as positron emission tomography and func-tional magnetic resonance imaging to advance our understanding of the higher brain processes that underlie pain.2 However, there are great limitations on the use of humans in experimental studies of pain and animal subjects continue to be vital. In vivo models are particularly important because pain and its underlying mecha-nisms are emergent processes of a whole nervous system; these processes cannot be fully simulated in highly reduced cell or tissue systems. In addition to bettering our understanding of noci-ception and pain, these models are valuable out of welfare con-cerns for achieving a better understanding of pain–nociception

processes in animals. In spite of great recent progress in decipher-ing the neurobiological basis of nociception and pain, this knowledge has yet to see large-scale translation into effective pain therapies. A limiting factor has been the often unsuccessful extrapolation from animal models to human clinical practice, as exemplifi ed by recent attempts to develop new pharmacological treatments for migraine headache.3–5 Benefi cial applications from animal models could be fostered by rigorous examination of the validity and limits of these models.3 This goal also hinges on a better understanding of the similarities and differences between nociception and pain in humans and in the animal models, an understanding that would make model selection and interpretation more valid for human applications.

This chapter’s principal objectives are to clarify distinctions between nociception and pain to improve the interpretation and validity of animal models and to discuss and evaluate commonly used and important models. There are several recent reviews, some highly detailed, on animal models in nociception–pain research that should be consulted by readers wishing further information.3,6–8

DEFINING PAIN IN HUMANS: IMPLICATIONS FOR ANIMAL MODELS

A valid working definition of pain is vital for efforts to explain its underlying mechanisms or develop therapeutic interventions. To this end, the International Association for the Study of Pain9

defi nes human pain as follows: (1) pain is an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage; (2) pain is always subjective; and (3) pain is sometimes reported in the absence of tissue damage and the definition of pain should avoid tying pain to an external eliciting stimulus. One of the most criti-cal conceptual advances in the understanding of pain is the dis-tinction between nociception and pain. As Wall10 emphasized, nociception that is “activity induced in the nociceptor and noci-ceptive pathways by a noxious stimulus is not pain, which is always a psychological state.” It is also critical to understand that the pain experience requires conscious awareness.11,12 In the usual course of events, tissue-damaging forms of stimuli excite nocicep-tors and this activity is conducted through peripheral nerves, the spinal cord, and subcortical brain structures to the cerebral cortex. If a person is conscious when nociception-related activity arrives in the cortex, further processing by extensive cortical regions results in pain.11–14

From: Sourcebook of Models for Biomedical Research(P. M. Conn, ed.), © 2008 Humana Press Inc., Totowa, NJ.

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The separateness of pain and nociception is seen in diverse ways. First, nociceptive processes do not always lead to pain. People can sustain severe injuries in warfare, sports, or everyday life and either not report pain or report it differently than the extent of an injury would suggest.15–17 Second, people with “functional” pain syndromes experience chronic, disturbing pain without any demonstrable tissue damage or pathology. Third, pain can be reduced by psychological manipulations such as a visual illusion18 or hypnotic suggestion.19 Fourth, pain has a strong social learning component and depends greatly on prior experience with it and interpersonal interactions that accompany this experience.13

THE NEUROLOGY OF NOCICEPTION AND PAIN There has been great progress in identifying the functional neuroanat-omy underlying nociception and pain. We will present a brief account of the neural structures and systems implicated in noci-ception and pain as it pertains to the use and interpretation of animal models. Numerous excellent reviews provide more detailed information.1,2,20–22

Studies of diverse mammals have shown that nociceptive stimuli activate two types of nociceptive receptors in body tissues: those that appear to respond exclusively to noxious mechanical, thermal, or chemical stimuli and those that respond to combina-tions of these stimuli (polymodal nociceptors). Activity is con-ducted from these receptors to the spinal cord through both myelinated (Aβ and Aδ) and unmyelinated (C) axons. These axons synapse on dorsal horn spinal neurons, principally in the more superficial laminae, where extensive processing occurs. Ascending projections arise from neurons in diverse dorsal horn laminae, principally lamina I and V, and travel through the con-tralateral lateral and ventral spinal pathways.

The ascending pathways have synaptic terminations in diverse regions of the brainstem, mainly catecholamine neuron groups, parabrachial nuclei of the pons, midbrain periaqueductal gray, and diverse sites through the brainstem reticular formation. The thala-mus receives multiple direct spinal projections to topographically organized lateral and nontopographically organized medial nuclei. There are significant differences between mammalian species at this level, particularly in the existence of a posterior ventromedial nucleus that seems to exist only in primates and is greatly enlarged in humans.21 Functional imaging studies in humans have consis-tently shown a diverse array of cortical structures to be activated specifi cally in association with perceived pain, including the fi rst and second somatosensory areas, anterior cingulate gyrus, insula, and prefrontal cortex. Diverse evidence indicates that the somato-sensory cortical zones are critical for the sensory-discriminative dimension of pain, that pain intensity is related to activation of multiple zones, especially involving both hemispheres, and that the emotional-evaluative (suffering) component depends on the anterior cingulate gyrus, insula, and prefrontal cortex. In addition, it is now well established that in humans, pain experience is absolutely dependent on the functioning of these neocortical and limbic cortical areas.22,23 The dependence of pain on these cortical regions makes sense also when it is considered that pain depends on the concurrent existence of another function: consciousness. Extensive evidence shows that the cortical regions known to be essential for pain greatly overlap with those vital to the existence of consciousness.11,14

In addition to the ascending pathways is a network of descend-ing modulatory controls, centered in the periaqueductal gray and

rostral ventromedial medulla, that exerts both antinociceptive and pronociceptive actions on ascending nociceptive signaling.24

THE ADAPTIVENESS OF NOCIFENSIVE BEHAV-IORS Nociceptors form a common underlying thread through-out the evolutionary history of multicellular organisms. Nociceptors have been observed in all bilaterally symmetrical multicellular organisms that have been examined, with the notable exception of elasmobranch fi shes.14 Even the leech has nociceptive neurons,25

many of which display close similarity to the polymodal nocicep-tor population that has been so well characterized in mammals.

Although nociceptors are common in the animal kingdom, the existence of nocifensive behaviors, the unconscious protective responses to noxious stimuli, is even more widespread and not specifi cally tied to possession of nociceptors.26 The single-celled paramecium (absent any possibility of a nervous system) exhibits protective responses to adverse environmental stimuli. Likewise, sponges with no nervous system and jellyfish with simple nerve nets have simple, but functional nocifensive behaviors. In these and more advanced bilaterally symmetrical invertebrate organ-isms as well as vertebrates, the nociceptive system and nocifen-sive behaviors constitute an essential component to survival. Importantly, the suite of responses to nociceptive stimuli does not end simply with withdrawal reflexes, but in advanced multicellu-lar organisms also includes complex arrays of endocrine and autonomic responses that help prepare the organism for a defense of disturbed homeostasis. Species with nociceptors showing prop-erties in common with those of mammals could serve as useful models for investigating peripheral nociception.

DISTINGUISHING NOCICEPTION FROM PAIN: WHY IT MATTERS As explained above, nociception and pain are distinctly different things, with differing underlying mechanisms. Unless one is studying the processes that specifically mediate the conscious experience of pain or a behavioral response that is specifi c to such processes, nociception is being studied and the term pain should not be used. Unfortunately, these terms are fre-quently used in ambiguous or inconsistent ways, with signifi cant practical costs in the use and interpretation of animal models and theoretical costs in understanding of mechanisms.

The Nociception–Pain Dichotomy in Clinical Neurol-ogy The behavioral separateness of nociception and nocifensive responses from pain is commonly seen in humans with severe neurological injury such as a spinal cord transection. Noxious stimulation of a limb below the level of the transection excites nociceptive sensory receptors and nociceptive pathways within the spinal cord. This spinal activity produces a nocifensive limb withdrawal response, but because nociceptive pathways are inter-rupted between the spinal cord and the cortex, no pain is felt. The pain–nociception distinction does not stop at this level.

Humans with massive damage or dysfunction of the cerebral cortex are unconscious, but can be awake and show grimacing, vocalization, and organized avoidance reactions in response to a nociceptive stimulus.12 The importance of understanding the noci-ception–pain distinction was shown by the confusion and conten-tiousness surrounding the recent tragic case of Terri Shaivo, who in 1990 experienced a prolonged period of anoxia. Although examining neurologists agreed that she was unconscious, in an irreversible, persistent vegetative state, Mrs. Shaivo was awake, was quite reactive to noxious stimuli, and exhibited nocifensive and emotion-like behaviors, which led to claims by some that Ms. Schaivo’s behaviors had to be consciously mediated. After a pro-

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tracted legal dispute and interventions by government offi cials, her feeding tube was removed. Subsequent autopsy confirmed that her cerebral cortex had massively degenerated, a fact consistent with the diagnosis of unconsciousness.28 The important points to be gleaned from this case are that a striking array of nocifensive behaviors can be unconsciously expressed in a human; that these are behaviors mediated by the brainstem and spinal systems rather than conscious, pain-related responses; and there is a widespread public unawareness of such things with associated societal consequences.

Valid Selection and Interpretation of Models As stated previously, effective therapies for pain have lagged behind advances in basic research. A practical example will serve to illustrate this point. Substance P was described in 1931 and its role in nociceptive neurotransmission has been known for decades.29 Immense resources have been devoted to development of substance P antagonists, principally NK1 receptor antagonists for pain relief, especially migraine headache. In spite of their effectiveness in animal models of dural inflammation, these drugs were ineffective in relieving human migraine pain. This negative result was not a matter of a receptor affinity mismatch because high level NK1 receptor blockade has been achieved in the brains of humans.30 This costly failure has been attributed to many things, including recognition that there is more to human migraine headache pain than events in the dura mater.4,31 Here, the animal model response measure was at the most peripheral level of the complex hierarchy of nociception–pain process, but the human clinical response measure, the pain report, was at the end stage of all intervening processing.

WHAT WOULD THE PAIN OF ANIMALS BE LIKE? A most important question regarding animal models for pain is which species are capable of pain experience, or at least a pain experience that meaningfully resembles that of a human. The foregoing explanation should make it clear that most species in the animal kingdom can detect and respond to noxious stimuli but not all can experience conscious pain. There has been interest in using invertebrates and some nonmammals such as amphibians32

as animal models for nociception on the assumption that they were more humane models than mammals. Examination of the collective neurological and behavioral evidence has led to the conclusion that fishes and amphibians are very unlikely to have a capacity for conscious pain experience, at least anything resem-bling that of humans.14,32 However, there are differing views.33,34

It is clear, though, that structural organization of the forebrain differs dramatically between mammals and nonmammalian ver-tebrates. Most notable is the unique presence of a neocortical component of the forebrain pallium in mammals,35,36 with its suite of structures, and especially interconnections between them, essential to pain experience. Nonetheless, the forebrain complex-ity of reptiles and birds is considerable36,37 and the presence of functional homologs of pain or consciousness-related cerebral structures, especially in birds, remains to be investigated.

The majority of animals used in nociception–pain research are mammals and the species used all have, to varying degrees, the cortical regions that correspond to those as essential to human pain experience.14,36 However, large expanses of higher–order nonsensory, nonmotor cortex are part of the pain-related mosaic and in nonprimate mammals, only a small proportion of cerebral cortex, less than 10% in rats and mice,38 fits this structural desig-nation. As mentioned previously, the subcortical pathways that

distribute nociception-related information to pain-related cortical zones also differ substantially between primates (especially humans) and nonprimate mammals. Clearly, significant functional differences may exist between species of mammals in general and humans in particular in terms of the how pain processing might occur.

WHEN IS AN ANIMAL MODEL APPROPRIATE FOR THE INVESTIGATION OF PAIN?

Much current research employs models specifically addressed at particular types of human pain conditions. Some models are also used to address fundamental mechanistic questions concern-ing nociceptive processing or actual pain experience. In either case, where an animal model is being used to investigate some aspect of human nociception and pain, it is vital to know that the model system is actually valid for the purpose. In particular, pain-related response measures must be distinguished from purely nociceptive–nocifensive response measures because pain is a psychological process that is not directly observable and must be measured indirectly through behavior.

As shown above, clinical neurology provides human examples of the pain–nociception distinction, but clear examples have been in the animal literature for many years. Responses to noxious stimuli have been studied in several mammalian species following decerebration where all of the forebrain above the midbrain is removed. Chronically decerebrate rats39 react strongly to the insertion of a feeding tube, struggling, pushing at it with the forepaws, and vocalizing. When receiving an injection, these rats react indistinguishably from a normal rat: vocalizing, attempting to bite the syringe or the experimenter’s hand and lick the injec-tion site. Since a large body of evidence indicates that conscious-ness (and accordingly pain experience) depends on the neocortex, it must be concluded that these reactions are nocifensive and unconscious rather than expressions of conscious pain. It can be concluded that nocifensive behaviors can be far more complex than “simple reflexes” and even ostensibly purposive, a fact that makes the behavioral distinction between nociception and pain diffi cult. In fact, many assumptions about indications of pain have been based on behaviors that are sustained, organized, or directed to the site of nociceptive stimulation,7,8,40 the type of responses fully within the capacity of decerebrate rats.

Identifi cation of pain in humans usually depends on a verbal report, but verbal reports and other pain-related behaviors are not always reliably interpretable.19 So, validated rating scales or other tools adopted from cognitive psychology provide sufficiently reli-able means of measuring pain in humans. Correspondingly, there is a long history in experimental psychology of using nonverbal behavioral methods to assess the internal “psychological” state of an animal. It is quite possible to assess the aversiveness of a stimulus in terms of whether the animal will learn to avoid the stimulus, escape from it, or perform some behavior to escape that refl ects the aversiveness of the nociceptive stimulus. An example of the last case is that a rat will leave a dark chamber and enter a brightly illuminated chamber (normally aversive to a rat) in order to escape a hot plate or electric shock.8 Learned avoidance, or conditioned emotional responses to nociceptive stimuli, however, does not prove the existence of conscious pain, because associa-tive learning of these types is believed to be unconsciously mediated.41

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Another effort to establish the existence of pain in an animal, as opposed to measurement of pain intensity, was Bateson’s40

eight criteria for animal pain, which have been of interest to welfare biologists. However, when viewed in terms of what is currently known about pain14 these criteria are flawed because they fail to distinguish nociception and nocifensive behaviors from conscious pain.34

EXAMPLES OF COMMONLY USED ANIMAL MODELS AND AN EVALUATION OF THEIR INTERPRETATION

Although overlap occurs in the use of some models, they can be broadly divided into those primarily used for investigation of acute nociception–pain and those for study of chronic nocicep-tion–pain. Because of the great clinical importance of chronic pain, more diverse and specialized models are used to represent particular human pathological processes such as neuropathic, bone cancer, or arthritis pain. In using waking animals for studies of nociception–pain, there is the ever-present issue of humane treatment.6,8 Investigators are bound by ethics and statute to mini-mize the exposure of animals to presumably painful procedures, within the objectives of a study. This requirement has shaped the development of the animal models for nociception–pain research toward the use of relatively low-intensity, brief-duration stimuli. The welfare consideration has been an impetus to develop in vitromodels or use species (such as amphibians; see Chapter 37 by Stevens in this volume) viewed as less likely to have a capacity for pain experience. Accordingly, as addressed below, these animal models must be validated for their intended application.

CONSTRUCTS AND TERMINOLOGY Nociception (and frequently pain) is a normal consequence of noxious stimulation, of course, but modifications of normal nociception–pain are more commonly of clinical importance. These include allodynia, hyper-algesia, analgesia, dyesthesia, paresthesia, causalgia, and neuro-pathic pain (Table 36–1). These terms represent human clinical complaints that animal models are intended to represent, but because some of these complaints, like paresthesia or causalgia, are identified by subjective reports, it is impossible to know how well the animal model represents them.

There are two main ways to categorize nociception/pain models1: (1) by stimulus duration, such as short duration (acute) stimuli and longer duration (chronic) stimuli, or (2) by level of the nervous system presumed to mediate the responses (Table 36–28). Ideally, a model should meet the following criteria: specifi city, sensitivity, construct validity, predictive validity, and reliability (Table 36–1).

The Commonly Used Models Contemporary models vary greatly in sophistication and validity but the predominantly used behavioral models employ rats or mice and are based on spinal refl exes such as tail flick and paw withdrawal and entail, vari-ously, measures of response threshold and/or latency. Additional measures associated with paw withdrawal are paw lifting, fl inch-ing, guarding, and licking. The most frequently used tests of nociception have been the tail flick, hot plate, paw pressure, writh-ing, and formalin tests.6,8 The tail flick test entails radiant heat application to a localized tail region or immersion in preheated water. Tail flick is known to be a spinal reflex, but is likely modu-lated by descending influences from the brainstem.6,8,24 A varia-tion on the tail flick test is the Hargreave’s test in which heat is

applied to the plantar surface of a foot and withdrawal latency is measured. In the hot plate test the animal is placed on a heated metallic plate and latencies for paw licking and jumping responses are recorded. These behaviors are mediated through a combina-tion of spinal and brainstem processes (Table 36–2). In the plantar pressure test, increasing force is applied on the plantar surface of a foot with a von Frey-type filament and threshold pressure for paw withdrawal is monitored. The latter two responses involve supraspinal, probably brainstem control (Table 36–2).

Electrical stimulation of the tail or paw has also been fre-quently used to elicit a hierarchy of responses, including twitch-ing, escape behavior, vocalization, and biting the electrodes, which are thought to reflect progressively more complex neural mediation. Dental pulp stimulation has also been used for the purpose of simulating trigeminal nociception–pain states and also

Table 36–1Constructs and terminology

Allodynia: pain caused by a normally innocuous stimulus.Analgesia: a consequence of a manipulation, such as drug

administration, that causes a previously pain-provoking stimulus to become nonpainful.

Causalgia: an abnormal spontaneous or stimulus-evoked burning sensation, generally due to neuropathic conditions.

Dyesthesia: an unpleasant abnormal sensation. Hyperalgesia: intensifi cation of the painfulness of a normally pain-

producing stimulus.Neuropathic pain: diverse pain experiences, including allodynia,

hyperalgesia, and spontaneous pain due to a nervous system lesion or disease.

Paresthesia: an abnormal spontaneous or stimulus-evoked sensation.

Specifi city: the stimuli used should be genuinely nociceptive. In practice, test stimuli may activate combinations of nociceptive and nonnociceptive sensory afferents. The behavioral response model should distinguish nonnociceptive from nociceptive stimuli.

Sensitivity: it should be possible to detect a range of responsiveness across a range of stimulus magnitudes, from below to above the nocifensive response threshold. Ideally, the model should also be sensitive to pharmacological or other manipulations that might modify responsiveness.

Construct validity: a most critical but often overlooked criterion is that the model should actually be an indication of nociception or pain. It is particularly important to know if the model is actually assessing pain as opposed to nociception alone. Put another way, the model should really be assessing the process or variable that it is thought to assess. If bone cancer pain in humans is of fundamental interest, the model must realistically replicate key pathophysiological and psychological attributes of human bone cancer pain. This issue emerges in many contexts, but is particularly salient in consideration of models that do not employ behavioral measures, such as histological or gene expression measures.

Predictive validity: effects of pharmacological or other therapies on the animal model should predict the effects of these treatments on human pain.

Reliability: The model must give the same results each time it is used, within and between laboratories, to make the results obtained adequately generalizable.

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on the (questionable6) assumption that all the associated afferents are nociceptors.

The commonly used formalin and writhing tests exemplify tests eliciting long-duration nociceptive stimulation by irritant injection. Intradermal formalin injections are most common, but hypertonic saline, complete Freund’s adjuvant, capsaicin, bee venoms, and other agents have been used. When injected into the dorsal surface of a rat’s forepaw, a 0.5–15% formalin solution evokes a constellation of quantifiable behaviors, including reduced weight bearing on the paw, paw lifting, licking, nibbling, shaking, or biting. Irritant injection into the footpad of rats or mice, in contrast, produces a biphasic response, with an initial phase of about 3 min latency, a subsequent period during which responses dissipate, and a later (20–30 min latency) reappearance of behav-ioral reactions, which appears to stem from predominant activa-tion of C fiber nociceptors.8

The writhing test involves intraperitoneal injection of any of several irritants, including phenylbenzoquinone, acetylcholine, dilute hydrochloric or acetic acid, and bradykinin. These sub-stances irritate serous membranes of the peritoneal cavity and in rats and mice evoke abdominal contractions, large body move-ments, including the hind paws, asymmetric contraction of dorsal abdominal muscles, and reduced locomotion. Although intended to be a model for visceral pain, it is likely that somatic afferents are also activated by the algogenic substances.

Models using stimulation of hollow organs, particularly gas-trointestinal or urogenital structures, have taken various forms: injection of formalin or other algogenic substances into the colon, capsaicin injection into the bladder, distention of organs like the colon and rectum with an inflatable balloon, or solid material introduction into a ureter. The behaviors produced depend to some extent on the specific type and location of the nociceptive stimulus, but include various types of abdominal reflexive con-tractions, stretching, and abdominal licking.

Models specifically designed to investigate chronic nocicep-tion–pain often include some of the same behavioral measures and nociceptive treatments previously described for investigations of acute nociception–pain. For example, the threshold nociceptive pressure required for paw withdrawal can be assessed in a normal rat by pressure with a filament (the von Frey test), but this method can test hyperalgesia after injection of an irritant like formalin into a paw. Likewise, this test can be used to assess allodynia associated with neuropathic pain caused by direct nerve injury or chronic nerve constriction.

Interpretations of the Commonly Used Behavioral Models The above-described tests are widely used because they

appear to have face validity, that is, they appear to produce states that would be comparable to those that would be associated with pain in humans. In addition, the most widely used of these, like the hot plate and intradermal formalin tests, are relatively easy to perform and score on large numbers of animals, thereby lending themselves to dose–effect studies of potentially analgesic drugs or other antinociceptive manipulations. The seeming simplicity of such tests is illusory. Detailed critiques of the limitations in these models have been presented by Le Bars et al.,6 Blackburn-Munro,3

and Vierck,8 and the interested reader is urged to read these infor-mative and thoughtful reviews.

A detailed discussion of the technical and theoretical limita-tions of the various nociception–pain models is not possible here, but we will outline the major concerns. The first such as noxious temperature paradigms (e.g., tail flick, hot plate, Hargreave’s) or paw withdrawal tests, there are many poorly controlled and vari-able aspects to nociceptive stimulus application. For example, a rat’s tail is a thermal exchange organ, which places it in a very different functional category from other tissues regarding effects of thermal stimuli.6 Furthermore, in the hot plate test, the animal’s movements make thermal stimulus application highly variable. Although nociceptive stimuli are often administered in ways intended to avoid or minimize tissue trauma, it may result in vari-able degrees, thereby introducing the dimension of allodynia or hyperalgesia in a test where these effects are not intended. Effects of injected algogenic stimuli will differ depending on the specifi c agent used (formalin, capsaicin, mustard oil, etc.) and route of administration (intraperitoneal, in the bladder) presenting a complex stimulus, to say the least. In addition, nonnociceptive afferent activation is often a confounding variable in that those afferents may contribute to the elicitation of responses that are presumed to be purely nocifensive.

Response measures themselves also present difficulties in the foregoing test paradigms.6,8 Investigator judgments are frequently a major source of variation in quantifying responses. Repeated testing of individual animals in some of the procedures, like the hot plate test, can be associated with learned aversion behaviors that modify stimulus application as well as the character of the responses. Sometimes tests of threshold become confounded with response latency, especially with longer duration stimuli. In addition, activation of nonnociceptive afferents in testing paradigms (above) may elicit flexion reflexes, confounding the interpretation of the nociceptive nature of the stimulus and response. Genetic variability and strain differences can also be a major source of confounding variability in a number of models.6,8

Table 36–2Common nociception testing paradigms,a observed behavioral outcomes, and their presumed neural substrates

Stimulus modality Testing paradigmsb Quantifi ed behaviors Minimal central substratesc

Mechanical Hargreave’s (von Frey) Withdrawal Segmental (spinal)Thermal Tail immersion (hot or cold), tail

fl ick, hot plate, cold plate, Hargreave’s (radiant heat)

Withdrawal, jumping, escape, licking, guarding

Segmental and suprasegmental (brainstem)

Chemical Formalin, acid, capsaicin, taxol Licking, biting, guarding, altered posture, writhing, vocalizing

Segmental and suprasegmental

aEmploying natural stimuli; nonnatural stimuli (e.g., electrical stimulation) are not included; see Le Bars et al.6bReviewed in Mogil et al.48

cReviewed in Vierck.8

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In most cases, the responses examined in the most frequently used tests such as tail flick, hot plate, paw withdrawal, and their variations could be entirely mediated by spinal reflexes or brain-stem–spinal motor programs, thus constituting unconscious noci-fensive responses (Table 36–28). Nonetheless, some higher brain infl uence is probably operating in an intact, awake animal, but its presence and nature are hard to separate from subcortical pro-cesses and it is also likely to be unconsciously mediated. Conse-quently, none of these tests can be legitimately viewed as tests of pain, since they do not depend on a consciously mediated response. In some cases, investigators are aware of this constraint and strictly adhere to the term nociception rather than pain in inter-preting their results. Unfortunately this is far from a universal practice and erroneous language and inference are common. Frequently “pain processing” or “pain transmission” is used to describe what is clearly nociceptive processing at the receptor, spinal, or subcortical level. Statements that can be found in the literature, such as pain in labor originates in the cervix or that there is inflammatory pain in the bladder, are mechanistically incorrect by implying that the conscious, psychological process of pain is somehow produced and experienced by peripheral tissues.

Development of well-validated models for pain, as opposed to nociception, is one of the most significant challenges to this fi eld. To this end, some investigators have utilized diverse paradigms requiring behaviors clearly beyond the realm of reflexes or complex, but probably unconsciously mediated motor programs. Behaviors that are more complex than stimulus-bound fl exion refl exes and are directed at the site of nociceptive stimulation, or sustained after stimulus termination, such as the vocalization afterdischarge, are sometimes taken to reflect pain. But recall that even decerebrate rats are capable of such behaviors. Measures involving learned behaviors, tasks where the animal can regulate nociceptive stimulus intensity, and assessments of disrupted feeding, sleep, or social behavior may have more validity as refl ecting pain.3,8 Presumably, these types of behaviors refl ect more complex, higher-order processing in the brain, but in the absence of rigorous validation, their acceptance as indications of pain should be considered tentative.

Interpretation of Nonbehavioral Measures of Nocicep-tion/Pain Numerous reduced preparations, even in vitro models, are being used for studies of nociception–pain. Tissue response measures, such as neuronal expression of the early oncogene c-fos42 and other markers of gene expression,43,44 are potentially associated with processes of importance for nociception, but their signifi cance must be qualified because they are quite removed from pain or even the dynamic, functional context of nociception. Expression of c-fos is commonly interpreted as an indication of neuronal response to sensory, including nociceptive stimuli. But unlike c-fos expression, neuronal responses to nociceptive stimuli are not all or none, not always excitatory, and not invariant. Rather, they may entail changes in pattern or inhibition, all of which are mechanistically important, but undetectable with markers such as c-fos. In addition, the proportion of neurons that expresses c-fos is often much smaller than the population that actually responds electrophysiologically42,45 and neurons in some neurophysiologically responsive brain regions do not express c-fos.42,45 There are additional problems with false-positive responses and dissociation between behavioral and c-fos responses to noci-ceptive stimuli.42 Clearly, these indirect measures, like behavioral

measures, require careful validation with respect to what they do or do not mean. Likewise, interpretations from response measures such as reflexes or neurophysiological recordings obtained under anesthesia are also subject to qualification. Measures of brain neuronal activity in animals or measures used in humans, such as functional magnetic resonance imaging, alone are never proof of pain. The relationship of such indicators to pain can be assessed only after pain has independently been shown to coexist with and be essential for putative pain-related properties of such neuro-physiological correlates. Furthermore, nothing short of an intact, fully functioning animal, particularly a mammal, is a potentially suitable model for investigations of human-like pain. Highly reduced preparations are primarily valuable for understanding some elements of nociceptive signaling or peripheral pathophysi-ological processes that might initiate nociceptive signaling.

RECOMMENDATIONS FOR MODEL SELECTION AND INTERPRETATION

This chapter has stressed the importance of critically examin-ing the validity of animal models to foster progress in understand-ing mechanisms of pain and the development of more effective treatments. To this end, we propose that particular consideration be given to the following questions: (1) is the problem or phe-nomenon of interest expressed at the level of nociceptive process-ing and nocifensive responses or the higher-order, end stage of pain; (2) is the response measure valid and appropriate for the question under investigation; (3) how valid are assumptions con-cerning the equivalence between the animal model and the human pain condition; and (4) is peripheral nociception of greatest importance, or is it essential to evaluate pain specifi cally?

The difficulty of knowing how well the putative pain experi-ence of an animal such as a rat or mouse translates to a human-like experience is likely to remain a limiting factor in development of models that validly assess the psychological experience of pain. Future success in development of pain therapies may often hinge on more exacting attention to this issue. There is a good deal of disagreement between investigators of animal psychology on which species might manifest consciousness, a prerequisite to pain experience as we know it,34,46 but there is a different approach to the question of pain. The dependence of the suffering dimension of human pain on cortical functioning, especially the cingulate gyrus, insula, and prefrontal cortex, is now well established.2,13,14,22,23 It is also commonly and probably safely assumed that similar cortical regions, where present, work in at least roughly similar ways across mammalian species. On this basis, it would be possible to provide a preliminary validation of a putative animal model for pain by showing that behaviors alleg-edly reflecting it depend on the functional integrity of these pain-mediating cortical zones. There would still be potential for misinterpretation by confusing nocifensive behaviors with pain-dependent behaviors, but by placing the control of the response measure at the same cortical regions known to be essential to pain experience in humans, the potential for examining common mech-anisms would be greatly facilitated. There is currently evidence that investigators of pain are using this approach.8,47

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