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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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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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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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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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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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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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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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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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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.
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