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1 Pain in Research Animals: General Principles and Considerations T his chapter presents an overview of the ethical, legal, and scientific reasons that mandate the alleviation of animal pain, drawing attention to the principles of the Three Rs (3Rs; replacement, refinement, and reduction) and the central role of refinement in the humane care and use of laboratory animals. It includes discussion of the fundamental concepts of the experience of pain and factors that affect pain aversiveness. It focuses on the potential causes of pain in research animals while broadly consider- ing evidence of pain in vertebrates. It concludes with a discussion of the particular circumstances that may justify pain in laboratory animals. WHY IS IT IMPORTANT TO RECOGNIZE AND ALLEVIATE ANIMAL PAIN? Most research using animals is for the direct or indirect benefit of society. Furthermore, most research on animals is funded, directly or indi- rectly, by the public. For both these reasons, the public has the right and responsibility to discuss how animal research is conducted. The public expects animal experimentation to be not only scientifically justifiable and valid but also humane, meaning that it results in minimal or no pain, stress, distress, or other negative impact on the welfare of the animals involved. When laboratory animals are subjected to conditions that do cause pain or distress, then ethically—at least from a utilitarian perspective—the benefits must outweigh the costs. This ethical justification depends on the challeng- ing balance between the benefits (primarily to humans) and the costs to experimental animals in the form of pain, distress, and euthanasia. 11

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12 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS These ethical expectations are embodied in the principles of the Three Rs: replacement, refinement, and reduction (the 3Rs; Russell and Burch 1959). As outlined in Appendix B, they are also enforced by laws and encouraged by professional guidelines. The 3Rs, formulated to protect the welfare of animals used in research, are widely accepted as interna- tional standards for the humane use of animals in research or testing. The National Centre for the Replacement, Refinement and Reduction of Ani- mals in Research (NC3Rs; http://www.nc3rs.org.uk) defines the Three Rs as follows: • Replacement refers to methods that replace or avoid the use of animals. Examples include the use of alternative methods (e.g., computer modeling, in vitro methods) or the replacement of higher- order animals such as mammals with “lower” animals (e.g., inver- tebrates, such as Drosophila and nematode worms). • Refinement refers to improvements to animal welfare in studies where the use of animals is unavoidable. Such improvements affect the lifetime experience of the animal and apply to husbandry or procedures that improve welfare and/or minimize pain, distress, lasting harm, or other threats to welfare. Examples of refinement include training animals to cooperate with certain procedures (e.g., blood sampling) to reduce stress, ensuring that accommodation meets animals’ needs (e.g., socially housing primates), and using appropriate anesthetic and analgesic drugs. The committee also urges the definition of humane endpoints for each experiment as an important refinement. • Reduction refers to methods that minimize animal use and enable researchers to obtain equivalent information from fewer animals or more information from the same number of animals. Such methods include appropriate experimental design, sample size determina- tion, statistical analysis, and the use of advanced noninvasive imag- ing techniques. The principle of refinement, especially in the context of animal pain, is central to many US regulations and guidelines (see Appendix B): almost all specify that procedures involving animals should (1) avoid or minimize discomfort and pain, and/or (2) otherwise include the provision of adequate pain relief unless the pain is justified scientifically. Minimizing animal pain whenever possible is thus important both ethi- cally and legally. It is also a practice that yields scientific and practical benefits, as discussed in Chapters 2 and 4. For example, the early experi- ence of pain in postnatal animals may lead to increased pain sensitivity in the insulted tissue later in life (Chapter 2), while effective pain management in all animals (Chapter 4) may improve healing rates, decrease mortality,

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13 PAIN IN RESEARCH ANIMALS and prevent the potentially confounding effects of untreated pain on many aspects of biological function (e.g., immune function, sleep, cognition, and many biological variables that are affected by stress; for discussion see Chapter 2). WHAT IS PAIN? Essential to any discussion of how to avoid or minimize pain in animals is a clear understanding and definition of pain and related terms. What exactly is pain? How does it differ from “nociception”? How does pain vary? And what dimensions of pain are most relevant to animal welfare? The International Association for the Study of Pain (IASP; www.iasp- pain.org) defines pain in humans as “an unpleasant sensory and emotional experience associated with actual or potential tissue damage, or described in terms of such damage” (IASP 1979). Pain typically involves a noxious stimulus or event that activates nociceptors in the body’s tissues that convey signals to the central nervous system, where they are processed and gener- ate multiple responses, including the “unpleasant sensory and emotional experience” central to the IASP definition. The anatomy and biology of pain are covered in more detail in Chapter 2. Some key issues and important terms are addressed below to highlight some of the challenges in under- standing animal pain. Noxious Stimuli and Nociception “Noxious stimuli” are events that damage or threaten damage to tissues (e.g., cutting, crushing, or burning stimuli) and that activate specialized sensory nerve endings called nociceptors. First described in the skin by Sherrington in 1906, nociceptors are also in muscle, joints, and viscera. Sherrington coined the term “nociception” to describe the detection of a noxious event by nociceptors. Nociception thus represents the peripheral and central nervous system processing of information about the internal or external environment as generated by nociceptor activation. This informa- tion is processed at both spinal and supraspinal levels of the central nervous system, providing details about the nature, intensity, location, and duration of noxious events. It is important to understand that stimuli adequate to activate nocicep- tors are not the same for all tissues; following are examples of common types of noxious stimuli for different tissues: • Skin: thermal (hot or cold), mechanical (cutting, pinching, crush- ing), and chemical (inflammatory and other mediators released from or synthesized by damaged skin, and exogenous chemical stimuli such as formalin, carrageenan, bee venom, capsaicin)

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14 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS • Joints: mechanical (rotation/torque beyond the joint’s normal range of motion) and chemical (inflammatory and other mediators released into or injected into the joint capsule) • Muscle: mechanical (blunt force, stretching, crushing, overuse) and chemical (inflammatory and other mediators released from or injected into muscle) • Viscera: mechanical (distension, traction on the mesentery) and chemical (inflammatory and other mediators released from inflamed or ischemic organs, inhaled irritants). Noxious stimulation triggers multiple physiological and behavioral responses, only one of which is the generation of the unpleasant emo- tional state of pain. Other behavioral and physiological responses include withdrawal reflexes, increases in heart rate and blood pressure, and other parameters. As discussed below (see Boxes 1-3 and 1-4), many of these responses can also occur in organisms that do not experience pain (e.g., anesthetized animals, or those with spinal lesions that prevent nociceptive information from reaching higher central nervous system structures). Thus pain and nociception are distinct concepts, and some nociceptive responses (e.g., withdrawal reflexes in spinal cord-transected animals) do not neces- sarily indicate pain. However, in the intact animal and in humans, noci- ceptive input reaches subcortical and cortical brain nuclei that contribute to the affective, aversive states of pain. In humans, therefore, nociceptive reflex withdrawal responses generally correlate with experiences of pain as evidenced by verbal feedback about the quality of the stimulus. Nonhuman animals cannot provide verbal feedback. Therefore, an ongoing challenge in laboratory animal research is to determine whether responses that could merely be nociceptive are also indicative of pain, and, conversely, whether the abolition of nociceptive responses indicates the successful abolition of pain. Thus, in the intact animal (e.g., under light anesthesia that removes some but not all responses to noxious stimuli), the distinction between nociception and pain is not always clear. Pain The generation of pain from nociceptive signals occurs in the central nervous system (CNS). Certain regions of the forebrain are responsible for the experience of both the sensory aspects of pain (i.e., qualitative properties such as location, duration, and whether “sharp” or “dull”) and the unpleasant, affective aspects associated with it (i.e., the way that pain “hurts”; Baliki et al. 2006; for details see Chapter 2). Studies of human pain have shown that pain is unpleasant and aversive: humans typically seek to avoid and minimize it. Furthermore, anticipation or threats of pain can cause anxiety and/or fear (Price 2002). This so-called “negative valence” of

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1 PAIN IN RESEARCH ANIMALS pain (i.e., the fact that it is aversive) underlies its description as emotional/ affective (Box 1-2). Aversiveness is thus a consistent characteristic of pain, but does not mean that all pain is the same: it varies in character (e.g., stinging, throbbing, aching, burning), location (e.g., joints, viscera), duration (from momentary to persistent or chronic), and intensity (from minimal to very intense). Pain can thus vary in its sensory, qualitative properties as well as in the extent of its aversiveness or unpleasantness. How aversive or unpleasant pain is depends primarily on its duration and intensity (Price 2002), although as explained below, psychological factors such as controllability can also affect the experience of pain. In terms of duration momentary pain is less aversive than persistent or chronic pain (see Box 1-1 for terminology and definitions). Indeed, many animals (and humans) are prepared to accept momentary discomfort or pain (e.g., that from a needle stick) especially if it is associated with a reward. In contrast, chronic pain (e.g., that caused by osteoarthritis or cancer) can be very difficult to manage and thus lead to distress and pathological changes that further undermine well-being (e.g., hypertension, immunosuppression, depression, cognitive changes, and possibly structural changes in the brain; Apkarian et al. 2004a,b). Similarly, intensity affects the aversiveness of pain. Intensity can vary from very low, when pain is first detected (the “pain threshold”), to the upper limit of tolerance and beyond (where tolerance is defined as the greatest intensity of pain that is accepted voluntarily). Obviously, more intense, severe pain is more aversive than slight pain. In humans, physiological and/or psychological state (e.g., stress, anxi- ety, fear) can also alter the aversiveness of pain (Carlsson et al. 2006; Keogh and Cochrane 2002; Price 2002). For example, pain that is controllable, predictable, or seen as ultimately yielding some benefit (e.g., the birth of a much-wanted child) is typically reported by humans as more tolerable and less aversive than uncontrollable, unpredictable pain of the same quality and intensity. Emerging evidence suggests that this may be true for some laboratory animals as well (Gentle 2001; Langford et al. 2006). Such factors are, how- ever, far less well understood for animals. Thus, efforts to alleviate pain in research animals typically focus on reducing its duration and/or intensity. Figure 1-1 helps illustrate how duration and intensity interact to affect aver- siveness. Indeed, the phrase “more than momentary or slight pain” appears repeatedly in animal protection legislation and guidelines1 (see Appendix B) to emphasize that longer-lasting or more intense pain should cause ethi- 1 For example, the duration and intensity of pain are central to USDA animal pain categories (where C refers to “minimal, transient, or no pain or distress,” and D and E procedures refer to “more than minimal or transient pain/or distress”; USDA 1997a).

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16 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS BOX 1-1 Terms Referring to the Duration of Pain The variety of terms used to describe the duration of pain can be imprecise and confusing, particularly because clinicians (e.g., veterinarians) and pain research- ers differ in their vocabulary. We present the terms here and explain how they are used in this report. Acute pain is used by pain researchers to refer to pain that is momentary, such as associated with a needle stick (e.g., drug injection, venipuncture) or an ex- perimentally applied noxious stimulus that does not produce noticeable tissue damage (e.g., pinch, mild electric shock). These experimental manipulations may generate a withdrawal reflex or vocalization. However, this pain is of very short duration (seconds to tens of seconds, perhaps minutes when assessing pain tolerance; see further discussion in text) and consequences to the subject are minimal and brief. In this document, momentary pain refers to this kind of brief, transient pain. Acute pain is also used in both human and animal clinical medicine to label the pain typically associated with procedures or surgery. Tissue injury is a usual consequence of such procedures and thus the pain induced is considerably lon- ger lasting than momentary (e.g., lasting for days to more than a week). In this document, pain of this nature is referred to as postprocedural or postsurgical pain.a Persistent pain refers in this report to pain states that can last for days or weeks but that are caused by different mechanisms than momentary or postprocedural pain.b To study these mechanisms numerous animal models have been developed that are commonly known as “persistent pain models.” These are described in Appendix A. Chronic pain, of long duration (weeks, months, or years), can be difficult to man- age in both human and animal clinical settings.c These pain states are distinct from postprocedural or persistent pain in that they are typically associated with tissue degenerative and destructive diseases (e.g., osteoarthritis, cancer) and do not improve or resolve over time. In the context of laboratory animal medicine, chronic pain is most commonly a byproduct of non-pain-related research (e.g., aging, disease research). aThe committee recognizes that the term “acute pain” is commonly used by human and animal clinicians/veterinarians to refer to postprocedural pain or “sharp” pain. However, “sharp” pain can be of both short or long duration (usually undefined), and “acute” means different things to different people. The committee therefore abstains from using the terms “acute” or “sharp” in favor of the terms “momentary” and “postprocedural” or “postsurgical” as defined above. bA common synonym for “persistent” is “tonic,” a description commonly used in pain re- search, that characterizes pain evoked for as long as nociceptors are stimulated. cChronic pain in humans is usually defined as pain lasting beyond the expected course of normal healing, often arbitrarily set at 6 months or beyond. Such duration is not appropriate to apply to laboratory animals with much shorter lifespans than humans or early developmental stages. Recurring or constant pain that lasts beyond the expected course of normal healing (which differs per species and per insult/injury) may merit consideration as “chronic pain.” The committee urges pain researchers, veterinarians, animal care staff, and IACUCs to recognize the influence of lifespan on the definition of chronic pain.

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1 PAIN IN RESEARCH ANIMALS FIGURE 1-1 The two key aspects of pain relevant to refinement. The aversiveness of pain (darker shading = greater aversiveness) is primarily determined by duration and intensity: momentary and/or slight pain is less aversive than chronic and/or intense pain. Duration and intensity interact to affect aversiveness, although not in a simple additive way (the shading on this diagram does not imply a linear relation- ship). In humans, the aversiveness of pain is also affected by psychological factors, such as how controllable or predictable the pain is, and its context or consequences. There is little information about the influence of such effects in other animals (but see Chapter 4); thus for most practical purposes, the alleviation of pain in research animals typically means reducing its duration and/or its intensity, and both are re- finements to be made whenever possible (see Chapters 3, 4, and 5). cal concern and serious consideration of its alleviation. Chapters 3 and 4 address this in more detail. An “unpleasant sensory and emotional experience” is at the core of the IASP definition of pain. Because sensory experiences and emotions (see Box 1-2) involve inner, private states that cannot be accessed directly by others, “[pain] is always subjective” (IASP 1979). This has some important practical implications. First, pain can never be measured directly, even when treating or researching human pain. Instead, the subjects’ reports of

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18 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS BOX 1-2 Emotion, Affect, Consciousness, and Awareness In everyday use, “emotion” means a feeling that is consciously experienced and either negative (e.g., fear) or positive (e.g., joy). To scientists specializing in emo- tion research, states that are positive (i.e., accepted/preferred) or negative (i.e., aversive/not tolerated/avoided) are said to have a property called “valence.” In the context of animal pain, the term “affect” instead of “emotion” is used because it is the scientific word whose meaning is closest to the colloquial use of “emotion,” while also being less anthropomorphic. Thus to scientists specializing in emotion research, “affect” (or “affective”) covers all states with valence; these include emo- tions (typically regarded as specific states induced by ongoing stimuli or events), moods (more generalized and longer lasting), and certain clinical conditions (e.g., depression) (Panksepp 2005; Rolls 2000, 2005; Russell 2003; Winkielman et al. 2005). However, some researchers use the terms specifically to refer to the hu- man experience of conscious feelings (Panksepp 2005; Russell 2003). This use of the terms “affect” and “affective” requires clarification of the terms “conscious” and “consciousness.” The word “conscious” has a range of mean- ings, from the experience of the most basic form of sensation to the ability to have higher-order thoughts about one’s own experiences, perspectives, or states of knowledge. In this report, conscious is used only to mean the former, thus referring to the “raw feel” of stimuli or events (Block 1997) or “the experience of sensation” (Merker 2007). Terms for this in specialized literatures include “qua- lia” (the inner “what it is like” aspects of, for example, seeing the color green or feeling angry; Tye 2007); “primary consciousness” (Edelman 2004); “qualitative consciousness” (van Gulick 2008); and “phenomenal consciousness” (Block 1997; Tye 2007). This basic form of consciousness is generally thought to be widely distributed across the animal kingdom (though how widely is a matter of debate; see text in this chapter). For brevity, in this report we use “consciousness” or “awareness” interchangeably. In the context of pain, such awareness is what distinguishes pain from nociception (see Boxes 1-3 and 1-4). their own pain (e.g., via verbal descriptions or Likert scale values) are used as proxy measures (see Chapter 3). Such a report is the closest we have to a “gold standard.” Second, in nonverbal organisms, be they laboratory animals or nonverbal humans such as babies, this type of self-report is not possible. As a result, making inferences about their pain is more challeng- ing. Box 1-3 defines some key terms central to understanding these complex and essential aspects of pain, and the following section discusses further key challenges in understanding and identifying animal pain. Box 1-4 outlines approaches that come closest to these “gold standards” in animal research: that is, the closest one can come experimentally to self-report in nonverbal subjects.

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1 PAIN IN RESEARCH ANIMALS BOX 1-3 Which (Unconscious) Nociceptive Responses May Not Indicate (Conscious) Pain? Various models and examples can help identify responses to noxious stimuli that do not necessarily involve pain. Such responses occur (1) in organisms with either no nervous system or a nervous system so simple that scientists believe the organism is not capable of affect; (2) in mammals whose forebrains are not receiving input from the periphery; and (3) in humans whose pain has been sup- pressed (e.g., by analgesics/anesthetics). Autonomic responses to noxious stimuli. In adult humans, postoperative cortisol output is undiminished by analgesics that successfully treat the reported pain (Schulze et al. 1988 cited by Lee et al. 2005; Dahl et al. 1992; see also Carrasco and Van de Kar 2003). Sympathetic responses such as tachycardia, hypertension, and pupil dilation occur in response to noxious stimuli in decerebrate rats and dogs (Sherrington 1906, reviewed in Sivarao et al. 2007). Simple avoidance responses. Nonlearned avoidance responses are present in even simple single-celled organisms and require no affect (Rolls 2000; Tye 2007; Winkielman et al. 2005). The withdrawal of body parts (e.g., limbs, tails) from noxious stimuli also occurs in decerebrate cats (Sherrington 1906), and spinal- transected cats and rats in which connections to the brain are severed (e.g., Grau et al. 1998). In spinally transected cats, pinching or clamping the tail promotes stepping movements of the hindlimbs (Lovely et al. 1986), as though simple locomotory escape movements can occur even without pain. Some learned avoidance responses (e.g., classically conditioned withdrawal) have even been observed in the sea slug Aplysia (reviewed by Allen 2004). Other research reveals the instrumental learning of avoidance responses normally associated with pain with no possible involvement of the brain: spinally transected rats learn to keep their limbs withdrawn for longer periods of time if doing so will terminate the insult (Grau et al. 1998). Other behavioral responses. Turning of the head and neck toward the noxious stimulus, some vocalization, and the licking of affected paws may occur in decer- ebrate animals (Baliki et al. 2005; King et al. 2003; Sherrington 1906). Other responses. Cerebral blood flow increases during venipuncture in human fetuses as young as 16 weeks gestational age, even though the thalamocortical connections required for nociceptive input to reach the cortex have not developed (Lee et al. 2005). Isoflurane-anesthetized rats show activation in several forebrain regions (e.g., cingulate and insular cortices) in response to noxious stimuli applied to a paw (Hess et al. 2007).a aThe responses listed here are unreliable as indices of pain when attemtpting to assess whether a particular species or stage of development can experience pain and not just nociception. To make this assessment, stronger evidence is required. The absence of this stronger evidence is what fuels debates about nonmammalian vertebrates (see Box 1-4 and text). In intact animals, however, these nociceptive responses do often play an important and significant role in pain assessment (see Chapter 3).

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20 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS BOX 1-4 Which Responses Indicate Pain and Which Nonhuman Vertebrates Display Them? To determine whether animals can experience pain (not simply nociception), it is neces- sary to show that they can discriminate painful from nonpainful states; make decisions based on this discrimination in a way that cannot arise from evolved nonconscious nociceptive responses (cf. text and Box 1-3); demonstrate motivations to avoid pain; and display affective states of fear or anxiety if threatened with noxious stimuli. In ad- dition, animals experiencing pain might be expected to exhibit spontaneous behavioral changes including sustained signals of distress and impairments in normal behaviors such as sleep (see text and Box 1-3). The discrimination of painful states: evidence from operant experiments. In some learn- ing paradigms, drug infusions are used as “discriminative stimuli,” that is, experimental cues that predict which of two alternative learned operant responses will yield reward (e.g., whether a right or a left lever press will deliver food). In such experiments, rats show by shifting their operant response for food that they are able to distinguish injec- tions of aspirin from injections of saline; furthermore, rats with arthritis learn this distinc- tion more readily than do control rats (Weissman 1976; see also Colpaert 1978 and Swedberg et al. 1988). Thus, pain can serve as a discriminative stimulus, something the committee does not believe could occur without awareness. Motivations to avoid pain or noxious stimuli. In learning paradigms in which an operant delivers an analgesic, rats in models-of-pain experiments lever press to self-medicate, and at a much higher rate than control animals. For example, rats with ligated spinal nerves lever press for clonidine, while controls do not (Martin et al. 2006). Rats, mice, primates, and pigeons also lever press to avoid electric shock (which may be painful depending on its intensity and duration; cf. Carlsson et al. 2006). Furthermore, oral self-administration of nonsteroidal anti-inflammatory drugs (NSAIDs) is observed in lame (i.e., arthritic) rats and chickens but not in their healthy counterparts (Colpaert et al. 1980; Danbury et al. 2000). Similar research has not been conducted on reptiles, amphibians, or fish but frogs, tadpoles, and fish do show conditioned active avoidance responses when a cue is paired with shock (Dunlop et al. 2003; Overmier and Papini 1986; Strickler-Shaw and Taylor 1991). Fish display this response even if it involves swimming over a hurdle that ANIMAL PAIN: DO ALL VERTEBRATES EXPERIENCE PAIN? The general acceptance that many animal species can experience pain underlies the emphasis on pain in guidelines and laws on humane care (see Appendix B) as well as the scientific validity of using animals to investigate clinical pain (see Appendix A). However, the question of which species and/or developmental stages experience pain, and which instead merely display nociception (cf. Boxes 1-2 and 1-3), is a complex and sometimes controversial topic. Some observers argue that only humans, specifically

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21 PAIN IN RESEARCH ANIMALS offers resistance (Behrend and Bitterman 1962). Similarly, fish learn to avoid hooks in angling trials (Beukema 1970). However, it is not certain that such simple avoidance learning requires the experience of conscious pain (see text and Box 1-3). Spontaneous behavioral changes. Noxious stimuli can cause vocalization (including ultrasonic calls in rodents) and signs of apparent apathy in mammals (see Chapter 3). Moreover, the use of inescapable electric shock to create mammal models of depres- sion is well documented in the neuroscience literature. Sleep disruption (assessed via EEG activity) occurs in rats with arthritis or persistent neuropathic pain (Blackburn- Munro 2004). Although these responses seem inconsistent with mere nociception (see Box 1-3), it is not yet proven that they result from pain. For instance, while fish injected with acid or bee venom show suppressed feeding and other behavioral alterations (Ash- ley et al. 2009; Sneddon et al. 2003a,b), such changes are not universally accepted as indicative of pain (Rose 2002). Recent studies with fish have shown, however, that the brain is active during noxious stimulation (with the forebrain being the most significantly affected) and that this activity differs from that of nonnoxious stimuli (Dunlop and Lam- ing 2005; Nordgreen et al. 2007; Reilly et al. 2008). In summary, evidence for the conscious experience of pain is strong for mammals and birds, but conclusive studies are either in progress for other taxa such as fish or have not yet been conducted. Pending such needed research, this report treats all verte- brates as capable of experiencing pain (see text).a aIt is important to remember that there is scientific evidence to suggest that pain or the threat of noxious stimuli causes fear and/or anxiety. Much research shows that the mere threat of foot shock (i.e., the application of electric current on the foot) induces behavioral and physiological signs of stress in rats and mice that can be alleviated with compounds that reduce anxiety in humans (anx- iolytics). Similar data are available for pigeons (Vanover et al. 2004). Furthermore, in one experiment an anxiety-inducing drug was used as a “discriminative stimulus” in pigs: the operant that would yield food was varied experimentally (e.g., from right lever to left lever) according to whether the subject was simultaneously infused with the anxiogenic drug or saline. Animals learned this discrimination successfully and performed a different operant for food depending on the compound of their infu- sion. The pigs were subsequently exposed to electric shock, which caused them to spontaneously select the anxiogenic rather than the saline operant when working for food. This finding suggests that the pigs’ experience of the electric shock included the sensation of anxiety (Carey and Fry 1993). No such research has been conducted on reptiles, amphibians, or fish. only humans past early infancy, experience pain (e.g., Carruthers 1996), while others suggest that all vertebrates, and some or even all invertebrates, are likely able to do so as well (Bateson 1991; Sherwin 2001; Tye 2007). Between these extremes lies a range of other, more generally accepted assessments. With a focus on vertebrates, this section presents a brief discussion of what constitutes good evidence of the capacity to experience pain. The dis- cussion emphasizes the strength of the evidence that all mammals (includ-

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22 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS ing rodents) are able to experience pain; raises the possibility that fish may feel pain; highlights the many things that are simply not known because the relevant research has not yet been conducted; and explains why the issue remains one of judgment rather than certainty. This section also lays the foundation for Chapter 3, on the recognition and assessment of pain. There are two broad methods of assessing which animals can experi- ence pain. The first is to demonstrate the presence of the anatomy and physiology that appear to be a requirement for pain in humans. The second is to investigate which species show responses to noxious stimuli suggestive of pain. Neither approach is adequate in itself, as noted below, but they are complementary and each informs the other. The anatomy and physiology of human pain are well understood: the nature of nociceptive inputs and circuits is well characterized, and specific forebrain regions (e.g., the insular, prefrontal, and anterior cingulate corti- ces) have been implicated in the experience of pain (see Baliki et al. 2006 and Chapter 2). Several authors have used this knowledge to catalogue similarities and differences between humans and other species (Allen 2004, 2006; Bateson 1991; Rose 2002; Sneddon 2006; Varner 1999). They typi- cally highlight homologies both in structure and in responses to noxious stimuli in the forebrains of humans and other mammals such as rats (see Apkarian et al. 2006; Borsook et al. 2006, 2007). Other vertebrates—birds, reptiles, fish, and amphibians—have peripheral and spinal nociceptive circuitry akin to that of humans, but not the specific forebrain regions involved in human pain. Invertebrates share still fewer similarities with humans—principally, only nociceptors and certain neurotransmitters (Allen 2004; Allen et al. 2005). The challenge in interpreting such data is knowing what emphasis to place on the various elements. Which, if any, underlie pain? Even the argument that certain forebrain structures are required for pain (Rose 2002) is problematic because it presupposes a complete understanding of how and where pain is generated in the human brain, when in fact this is still under study (the anterior cingulate, for instance, is activated by subliminal stimuli—i.e., stimuli of which humans are unaware—as well as by pain; Kilgore and Yurgelun-Todd 2004; Sidhu et al. 2004; Box 1-3). Such an argu- ment also assumes that, evolutionarily, any cortical subregions involved in pain became so only after their specialization into these subregions (thus ignoring the possible functions of these regions’ evolutionary precursors). Furthermore, it does not clarify the states of animals whose nervous systems differ greatly from that of humans but may still have analogous structures and functions (e.g., invertebrates, which lack a central nervous system, and birds or fish, which have complex forebrains but no neocortex; Allen 2004; Shriver 2006). This type of uncertainty is one reason the phylogenetic dis- tribution of pain is a matter of discussion and debate.

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23 PAIN IN RESEARCH ANIMALS Despite these ongoing debates, it is generally agreed that, in mam- mals, pain does require a cortex (though see Merker 2007 for an opposing view). Therefore, it is typically assumed that any responses in, for example, decerebrate mammals cannot be used reliably to identify which species or developmental stages feel pain (Box 1-3). The second way to determine which animals experience pain is by examining their physiological and behavioral responses to noxious stimuli. Pain in humans is associated with a range of physiological and behav- ioral responses. Some are best described as nociceptive because they occur in response to noxious stimuli even when pain is suppressed by analgesia or anesthesia (Box 1-3). But humans can also assess and report the presence or absence of pain, describe its qualities, and use this information to make decisions (e.g., when to seek help, when to take analgesics, or which pain management strategy to adopt). Pain also leads to the protection and “nurs- ing” of affected regions. Such behaviors reflect a strong, sustained desire to minimize or end pain (it has been argued that the affective component of pain is essential for the way it strongly motivates escape and avoidance; van Gulick 2008; McMillan 2003).2 As recent studies have demonstrated, postsurgical/postprocedural, persistent, or chronic pain can have deleteri- ous effects on behavior, cognition, and brain function (e.g., problems with sleep, attention, or depression, even possible loss of gray matter; Apkarian et al. 2004a,b). These findings suggest several useful indices for identifying animals that experience pain, not simply nociception (Box 1-4). Unfortu- nately, data on these key variables for many animal species have not been collected, generally because the research is methodologically challenging (Box 1-4). This is another reason why the phylogenetic distribution of pain is a matter of discussion and debate. Although definitive evidence is often unavailable, this report does not treat the absence of evidence as evidence of absence. Instead, the consen- sus of the committee is that all vertebrates should be considered capable of experiencing pain. This judgment is based on the following two premises: (1) the strong likelihood that this is correct, particularly for mammals and birds (Box 1-4 provides compelling evidence for rats, for example); and (2) the consequences of being wrong, that is, acting on the assumption that all vertebrates are not able to experience pain and so treating pain as though it were merely nociception, an error with obvious and serious ethi- cal implications. This report, therefore, considers nociceptive responses in vertebrates as likely indices of pain rather than nonconscious responses to noxious stimuli. 2 Asexplained in Chapter 4, the protective role of pain is one reason that complete elimina- tion of postoperative pain may not be desirable.

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24 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS CAUSES OF PAIN IN RESEARCH ANIMALS Understanding the potential causes of pain in research animals can facilitate the anticipation or recognition of both the types of specific stimuli or tissue responses and the situations (in terms of management, husbandry, or experiment) in which pain is likely. As a general guideline to types of stimuli or tissue responses that cause pain in animals, many codes and recommendations state something like the following: “Unless the contrary is established, investigators should consider that procedures that cause pain or distress in human beings may cause pain or distress in other animals” (Principle #4, US Government Principles for the Utilization and Care of Vertebrate Animals Used in Testing, Research, and Teaching; IRAC 1985); or “[a painful procedure is] any procedure that would reasonably be expected to cause more than slight or momentary pain and/or distress in a human being to which the procedure is applied” (USDA Policy #11; see Kohn et al. 2007 for a similar view from the American Col- lege of Laboratory Animal Medicine). The committee agrees with these statements, but cautions that in humans the type and intensity of stimuli detected by nociceptors differ for different tissues (as outlined previously in this chapter). For example, cut- ting, crushing, or burning skin reliably causes pain, whereas these same stimuli applied to the wall of a hollow organ rarely cause pain (see Ness and Gebhart 1990 for a review). If this is true for a single species, it is not hard to imagine the differences that may exist across the tissues of different spe- cies, especially those that have evolved to live in very different worlds (e.g., very hot or cold environments) or to have very different sensory abilities (e.g., abilities to detect ultrasound or electromagnetic fields; Allen 2004). Indeed, species-specific differences in response to painful events are well documented (Paul-Murphy et al. 2004; Valverde and Gunkel 2005). There is also variation in response to drugs that are analgesic in one species but not in another; for example, the effects of opioids are very unpredictable in birds (Hughes and Sufka 1991). For all these reasons, one cannot assume that what causes pain in humans will do so in all other organisms, and conversely, that what does not cause humans pain is equally benign in all other organisms. Thus it is essential to assess pain in an animal on a case- by-case basis (see Chapter 3). Examples of stimuli or tissue injury that cause pain in research animals, whether from disease conditions or experimental procedures, are given in Table 1-1. They are broadly broken down by tissue type, to mirror the tissue- specific noxious stimuli listed in the section above on nociception. The list in Table 1-1 is intended to be illustrative, not all-inclusive. Note that when assessed using the techniques discussed in Chapter 3, the aversiveness of the pain resulting from each item in the table can vary greatly (typically

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2 PAIN IN RESEARCH ANIMALS TABLE 1-1 Examples of Painful Procedures or Conditions by Type and Anatomic Location Abdominal Peritonitis, pancreatitis, hepatitis, cholelithiasis, distension of viscera, bowel obstruction, visceral tumors, laparotomy Cardiothoracic Myocarditis, pneumonitis, myocardial infarction, pneumonia, bronchitis, vasculitis, vascular grafts, thoracotomy Dermatologic Pruritis, chemical and thermal burns, cellulitis, otitis, skin tumors, incision, needle puncture Musculoskeletal Restraint, arthritis, periostitis, ischemia, application of a tourniquet, tendonitis, inflammation of joints, deep chemical or thermal burns, crush, bruising, necrosis, fracture, bone graft harvest, bone tumor, osteotomy, incision, craniectomy, degenerative joint disease Neurologic Encephalitis, meningitis; crush, ligation, or transection of nerves; tumor of neural tissue; neuroma Ocular Glaucoma, uveitis, corneal ulcer, orbital blood sampling, ocular tumor Orofacial Oral tumors, temporomandibular joint disease, gingivitis, tooth extraction, pulpotomy, tooth abscess Systemic Sepsis, sickness syndrome, autoimmune diseases Urogenital Pyelonephritis, cystitis, acute renal failure, ureteral or urethral obstruction, pyometra, urinary catheterization, mastitis, ovariohysterectomy, castration, urogenital tumor, dystocia from mild to severe), depending on its duration and intensity. Again, case- by-case assessment and treatment are critical and essential (see Chapters 3 and 4). In the context of animals used in research and testing, the following circumstances will or are likely to cause pain3: Non-research-related disease or injury: Tissue damage and/or inflammation (e.g., injuries sustained in fighting with conspecifics, ammonia burns from soiled litter), mastitis, abscesses and other infections, arthritis and other diseases resulting from aging, and parturition. Husbandry or eterinary treatment: Invasive procedures as part of normal husbandry, preparation for research, or before the animal’s designation as a research subject (e.g., castration, dehorning, teeth clipping, tail docking, tail-tip removal for genotyping, ear notching, microchip implantation, cath- eter placement, injection). 3 It is important to remember that early postnatal tissue injury can alter adult nociceptive processing, including enhanced responses to noxious stimuli.

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26 RECOGNITION AND ALLEVIATION OF PAIN IN LABORATORY ANIMALS Research byproduct: Research on disease (infectious or noninfectious, such as cancer), toxins, tissue damage (e.g., burns, bone breakage), some aspects of drug dependence (e.g., opiate withdrawal that causes lower back and/or abdominal pain and cramps); and surgery, in which pain may be a conse- quence of research but is neither an element of the research nor a focus of study. Hyperalgesia may also occur as a result of “sickness syndrome” (see Chapter 4). The use of pain as a tool to motiate or shape behaior: Noxious stimuli (e.g., foot shock) for the purposes of training or motivation during behav- ioral experiments (punishment/negative reinforcement), for the experimental assessment of fear (e.g., in fear-conditioning paradigms), or for the experi- mental induction of depression-like states. Pain as the focus of research: For a review and description of common animal models of persistent pain, including humane endpoints for this type of research, see Appendix A. These five circumstances may involve pain that differs in causation, duration, and intensity. They also vary in the nature and defensibility of the justification for inducing that pain and for allowing it to be untreated, as discussed below. IS PAIN IN ANIMALS EVER JUSTIFIABLE? According to current US laws and guidelines, some animal pain is justi- fied in some circumstances. For example, USDA Policy #12 states that “a description of procedures or methods designed to assure that discomfort and pain to animals will be limited to that which is unavoidable in the conduct of scientifically valuable research” (USDA 1997b), the Public Health Ser- vice Policy on Humane Care and Use of Laboratory Animals (DHHS 2002) mandates that “procedures which may cause more than momentary or slight pain or distress to animals should be performed with appropriate sedation, analgesia, or anesthesia, unless the procedure is justified for scientific rea- sons in writing by the investigator,” and section 2.31(e) of the US Animal Welfare Act states that “A description of procedures designed to assure that discomfort and pain to animals will be limited to that which is unavoidable for the conduct of scientifically valuable research” (AWA 1990). Thus there exist situations in which pain and/or the withholding of analgesic drugs can be justified scientifically. As noted above, such situ- ations include the use of noxious stimuli as a tool to motivate or shape behavior or the study of pain as the focus of research (see Appendix A). However, as indicated at the beginning of this chapter, the ethical justifica-

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2 PAIN IN RESEARCH ANIMALS tion for such research should consider both the costs to the animal and the expected benefits of the research to humankind (although a small research component may directly benefit the animals themselves, for example, in the development of better analgesics for rats or mice; for an in-depth ethical analysis, see “Animal Welfare Considerations of Research with Persistent Pain Models” in Chapter 4). Consistent with the concerns of the general public (Kohn et al. 2007) it is the view of this committee that the greater the cost to the research animals in terms of pain, distress, and negative impact on welfare and well-being, the stronger the scientific justification of the research should be. CONCLUSIONS AND RECOMMENDATIONS 1. Although there is general agreement that pain is an aversive state experienced by mammals and probably all vertebrates, the com- mittee assumes in this report that all vertebrates are capable of experiencing pain. 2. The assumption of similarities in pain between humans and ani- mals is a useful rule of thumb. However, the scientific outcomes should be taken into account when the 4th Government Principle is interpreted. 3. Pain in research animals may be induced deliberately as part of a research procedure (e.g., when pain is the subject of research) or may be an unintended byproduct of other research objectives, husbandry, or other factors. 4. As was emphasized in the Distress report (NRC 2008), the Three Rs (replacement, refinement, and reduction) should be the stan- dard for identifying, modifying, avoiding, and minimizing most causes of pain in laboratory animals. While research on pain or on methods of alleviating pain may unavoidably cause animal distress and severe perturbation of animal welfare, the goal of researchers, veterinary teams, and IACUCs should be to reduce and alleviate pain in laboratory animals to the minimum necessary to achieve the scientific objective. REFERENCES Allen C. 2004. Animal pain. Noûs 38:617-643. Allen C. 2006. Animal consciousness. The Stanford Encyclopedia of Philosophy (Winter Edition). Available at http://plato.stanford.edu/archives/win2006/entries/consciousness- animal/. Accessed June 3, 2008. Allen C, Fuchs PN, Shriver A, Wilson HD. 2005. Deciphering animal pain. In: Ayded M, ed. Pain: New Essays on Its Nature and the Methodology of Its Study. Cambridge, MA: MIT Press.

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