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CHAPTER 1. Pain in research animals: General
principles and considerations
This 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 that the principle of refinement plays in the humane care and
use of laboratory animals. It discusses 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 considering evidence of pain in
vertebrates. Finally, it concludes with a discussion of the justification of 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 and carried out in its name. Furthermore, most research on animals is
funded, directly or indirectly, 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 to be humane, meaning that it is undertaken with
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 challenging balance between the benefits (almost exclusively
to humans) and the costs to experimental animals in the form of pain, distress,
and euthanasia.
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 2, they are also enforced and encouraged by laws and
professional guidelines. The 3Rs, formulated to protect the welfare of animals
used in research, are widely accepted as international standards for the
humane use of animals in research or testing. The National Centre for the
Replacement, Refinement and Reduction of Animals in Research (NC3Rs;
http://www.nc3rs.org.uk) defines the Three Rs as:
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12 Recognition and Alleviation of Pain in Laboratory Animals
Replacement refers to methods that replace or avoid the use of animals.
Examples include the use of alternative methodologies (e.g., computer
modeling, in vitro methods, etc.) or the replacement of higher-order animals
such as mammals with “lower” animals (e.g., invertebrates, such as Drosophila
and nematode worms).
Refinement refers to improvements to animal welfare in situations
where the use of animals is unavoidable. Such improvements affect the
lifetime experience of the animal, 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 suggests
that defining humane endpoints for each experiment is 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. Examples include appropriate
experimental design, sample size determination, and statistical analysis, and
the use of advanced non-invasive imaging techniques.
The principle of refinement, especially in the context of animal pain, is
central to many U.S. regulations and guidelines. Almost all regulations and
policies (see Appendix 2) specify that procedures involving animals should avoid
or minimize discomfort and pain, and that adequate pain relief be provided
unless justified scientifically.
Minimizing animal pain, wherever possible, is thus important both
ethically and legally. It is also a practice that yields scientific and practical
benefits as discussed in Chapters 2 and 4. For example, the early experience
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, and avoid
the potentially confounding effects that untreated pain can have 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 “pain” differ from “nociception”? How does pain vary? And
which dimensions of pain are most relevant to animal welfare?
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CHAPTER 1: PAIN IN RESEARCH ANIMALS 13
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 located in the body’s tissues that convey
signals to the central nervous system, where they are processed and generate
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 faced in understanding
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 found in muscle, joints, and viscera.
Sherrington introduced the concept of “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 information
is processed at both spinal and supraspinal levels of the central nervous
system, providing details about the quality, intensity, location, and duration of
noxious events.
It is important to understand that stimuli adequate to activate
nociceptors are not the same for all tissues; following are examples of common
noxious stimuli in different tissues:
Skin: thermal (hot or cold), mechanical (cutting, pinching, crushing),
and chemical (inflammatory and other mediators released from or synthesized
by damaged skin, and exogenous chemical stimuli such as formalin, carragenan,
bee venom, capsaicin).
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, stretch, crush, 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 emotional
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14 Recognition and Alleviation of Pain in Laboratory Animals
state of pain. Other behavioral and physiological responses include withdrawal
reflexes, increases in heartrate, blood pressure and other parameters. As
discussed below (see Boxes 1-3 and 1-4), many of these latter responses can
also be seen 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 necessarily indicate pain.
However, in the intact animal and in humans, nociceptive 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 the fact that
humans provide verbal feedback about the quality of the stimulus. Non-human
animals cannot provide verbal feedback this way. Therefore, it is an ongoing
challenge in laboratory animal research 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 of or threats of pain can cause anxiety and/or
fear (Price 2002). This so-called “negative valence” of pain (i.e., the fact that
it is aversive) underlies its description as emotional/affective (see Box 1-1 for
further definition).
Aversiveness is thus a consistent characteristic of pain, but this does not
mean that all pain is the same: it varies in character (e.g., stinging, throbbing,
aching, burning); source (e.g., joints, viscera); and 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 a pain is depends
primarily on its duration and intensity (Price 2002), although as explained
further below, psychological factors such as controllability can also affect the
experience of pain.
Thus, 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
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CHAPTER 1: PAIN IN RESEARCH ANIMALS 15
discomfort or pain (e.g., that from a needle stick) in order to obtain rewards.
In contrast, chronic pain (e.g., that caused by osteoarthritis or cancer) can
lead to distress, can be very difficult to manage, and can lead to 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. Intensities can vary
from very low, where 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.
BOX 1-1 Terms referring to the duration of pain
A variety of terms describe the duration of pain and they can be
imprecise and confusing, particularly because clinicians (e.g., veterinarians)
and pain researchers 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 experimentally 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 is used to identify this kind of brief, transient pain.
However, 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 longer lasting than momentary (e.g., lasting for days to
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16 Recognition and Alleviation of Pain in Laboratory Animals
more than a week). In this document, pain of this nature is referred to as
post-procedural or post-surgical pain.2
Persistent pain is another descriptor used in this document to refer to
pain states that can last for days to weeks but that are caused by different
mechanisms than momentary or post-procedural pain3. To study these
mechanisms numerous animal models have been developed that are commonly
known as “persistent pain models”. These are discussed in Appendix 1.
Chronic pain, commonly used to describe pain of long duration (weeks,
months, or years), can be difficult to manage in both human and animal clinical
settings4. These pain states are distinct from post-procedural 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)
In humans, physiological and/or psychological state (e.g., stress,
anxiety, 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
2
The Committee recognizes that the term “acute pain” is commonly used by human and animal
clinicians/veterinarians to refer to post-procedural pain or “sharp” pain. However, “sharp”
pain can be both of 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 “post-procedural” or “post-surgical” as
defined above.
3
A common synonym for “persistent” is “tonic”, a description commonly used in pain research,
that characterizes pain evoked for as long as nociceptors are stimulated.
Chronic 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 concideration 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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CHAPTER 1: PAIN IN RESEARCH ANIMALS 17
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,
however, 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
aversiveness. Indeed, the phrase “more than momentary or slight pain”
appears repeatedly in animal protection legislation and guidelines5 (see
Appendix 2) to emphasize that longer-lasting or more intense pain should cause
ethical concern and its alleviation must be taken seriously. 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, meaning, that “[pain] is always subjective” (IASP 1979).
This is because sensory experiences and emotions (see Box 1-2) involve inner,
private states that cannot be accessed directly by others. This has some
important practical implications. Pain can never be measured directly, even
when treating or researching human pain. Instead, the subjects’ reports of
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”. In nonverbal organisms, be they laboratory animals, or non-
verbal humans such as babies, we cannot use this type of self-report. As a
result, making inferences about their pain is more challenging. Box 1-2 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 1-3 outlines approaches that
come closest to these ‘gold standards’ in animal research: i.e., the closest one
can come experimentally to self-report in nonverbal subjects.
5
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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18 Recognition and Alleviation of Pain in Laboratory 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 relationship). In humans, the
aversiveness of pain is also affected by additional 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 refinements to be made whenever possible (see
Chapters 3, 4 and 5).
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CHAPTER 1: PAIN IN RESEARCH ANIMALS 19
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 emotion 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 emotions
(typically regarded as specific states induced by on-going 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). Some researchers use the terms specifically to refer to the human
experience of conscious feelings (Panksepp 2005; Russell 2003). It is in this
latter sense that the terms “affect” and “affective” are used in this report.
This use of the terms “affect” and “affective” requires clarification of
the terms “consciously” and “consciousness”. The word “conscious” has a
range of meanings, 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 et
al. 1997) or “the experience of sensation” (Merker 2007). Terms for this in
specialized literatures include ”qualia” (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 et al 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 simply 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).
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20 Recognition and Alleviation of Pain in Laboratory 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 suppressed 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 der 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. Non-learned 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 in cat and rat spinal-transected preparations 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 also
occur even without pain. Some learned avoidance responses (e.g., classically
conditioned withdrawal) have even been observed in the seaslug 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 be
seen in decerebrate 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
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CHAPTER 1: PAIN IN RESEARCH ANIMALS 21
have not developed (Lee et al. 2005). Activation in several forebrain regions
(e.g., cingulate and insular cortices) in response to noxious stimuli applied to a
paw is seen in isoflurane-anesthetized rats (Hess et al. 2007).6
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 2) as well as the scientific validity of using animals to investigate
clinical pain (see Appendix 1). 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 argue that only humans, specifically only humans past early
infancy, experience pain (e.g., Carruthers 1996) hile 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 lie a range
of other, more generally accepted assessments.
With a focus on vertebrates, this section briefly considers what
constitutes good evidence of the capacity to experience pain. The discussion
emphasizes the strength of the evidence that all mammals [including 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.
There are two broad methods of assessing which animals can experience
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.
6
The responses listed here are unreliable as indices of pain when attempting 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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24 Recognition and Alleviation of Pain in Laboratory Animals
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 learning paradigms, drug infusions are used as
‘discriminative stimuli’, i.e., 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 the operant response they make for food that they are able to
distinguish injections of aspirin from injections of saline; furthermore, rats
with arthritis learn this distinction more readily than do control rats (Weissman
1976; see also Colpaert 1978 and Swedberg et al. 1988). Thus, pain can be
used 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 will
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 will
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 non-steroidal 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.
Frogs, tadpoles, and fish do show conditioned active avoidance responses when
a cue is paired with shock (Dunlop et al. 2003; Overmeier and Papini 1986;
Strickler-Shaw and Taylor 1991). Fish display this response even if it involves
swimming over a hurdle that 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 depression is well documented in the neuroscience
literature. Sleep disruption (assessed via EEG activity) is also observed in rats
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CHAPTER 1: PAIN IN RESEARCH ANIMALS 25
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 (Ashley et al 2009; Sneddon et al. 2003a,b) such changes are not
universally accepted as pain related (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 non-noxious stimuli (Dunlop and Laming 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 such conclusive studies are either currently being
undertaken for other taxa such as fish or have not yet been conducted.
Pending such needed research, this report treats all vertebrates as capable of
experiencing pain (see text).8
Causes of pain in research animals
Understanding the potential causes of pain in research animals can
facilitae the anticipation or recognition of both the types of specific stimuli or
tissue responses in which pain is likely and the situations (in terms of
management, husbandry, or experiment) in which pain is likely.
8
It is important to remember that there is scientific evidence to suggest that pain or
the threat of noxious stimuli cause 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 (anxiolytics). Similar data are available for pigeons (Vanover et al.
1994). Furthermore, in one experiment an anxiety-inducing drug was used as a ‘discriminative
stimulus’ (see Box 1-4 above) in pigs; thus the operant that would yield food was varied
experimentally (e.g., from right-lever to left-lever) according whether the subject was
simultaneously infused with the anxiogenic drug or saline. Animals learned this discrimination
successfully, i.e., they would perform a different operant for food depending on the compound
they were currently being infused with. Subsequently, the pigs were 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.
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26 Recognition and Alleviation of Pain in Laboratory Animals
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, U.S. 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 ACLAM).
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, cutting,
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 within a single species, it is not hard to
imagine the differences that may exist across the tissues of different species,
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
that the assessment of pain in an animal be done on a case-by-case basis (see
Chapter 3).
Examples of stimuli or tissue injury that cause pain in research animals
are given in Table 1-1. These may arise from a variety of disease conditions or
experimental procedures. In this table, they are broadly broken down by tissue
type, to mirror the tissue-specific noxious stimuli listed earlier in the section
on nociception. The list presented 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 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).
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CHAPTER 1: PAIN IN RESEARCH ANIMALS 27
Table 1-1 Examples of painful procedures or conditions by type
or 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
Facial Oral tumors, temporomandibular joint disease, gingivitis, tooth extraction,
pulpotomy, tooth abscess
Musculoskeletal Restraint, arthritis, periostitis, ischemia, application of a tourniquet,
tendonitis, inflammation of joints, deep chemical and thermal burns, crush,
bruising, necrosis, fracture, bone graft harvest, bone tumor, osteotomy,
incision, craniectomy, degenerative joint disease
Neurologic Encephalitis, meningitis; crush, ligation, and transection of nerves; tumor of
neural tissue; neuroma
Ocular Glaucoma, uveitis, corneal ulcer, orbital blood sampling, ocular tumor
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
In the context of animals used in research and testing, the following
circumstances will or are likely to cause pain9:
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 diseases resulting from aging, parturition.
9
It is important to remember that early post-natal tissue injury can alter adult nociceptive
processing, including enhanced responses to noxious stimuli.
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28 Recognition and Alleviation of Pain in Laboratory Animals
Husbandry or veterinary treatment: Invasive procedures as part of
normal husbandry, preparation for research, or even before becoming research
subjects (e.g., castration, dehorning, teeth -clipping, tail-docking, tail-tip
removal for genotyping, ear-notching, microchip implantation, catheter
placement, injection).
Research by-product: Research on disease (infectious, or non-infectious,
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
consequence 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 motivate or “shape” behavior: Noxious
stimuli (e.g., footshock) for the purposes of training or motivation during
behavioral experiments (punishment/negative reinforcement), for the
experimental assessment of fear (e.g. in fear-conditioingn paradigms), or for
the experimental 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 of this type of
research study see Appendix A.
These five circumstances may involve pain that differs in causation,
duration, and intensity. They also vary in the nature and strength 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 laws and guidelines, some animal pain is justified in
some circumstances. For example, USDA Policy #12 states 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 Service
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 reasons in writing
by the investigator”, and section 2.31(e) of the U.S. Animal Welfare Act states
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 situations
include the use of noxious stimuli as a tool to motivate or shape behavior or
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CHAPTER 1: PAIN IN RESEARCH ANIMALS 29
the study of pain as the focus of research (see Appendix 1). However, as
indicated at the beginning of this chapter, the ethical justification 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, 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 animal welfare and well-being,
the stronger the scientific justification of the research should be.
Conclusions and recommendations
1. Pain is an aversive state experienced by mammals and probably all
vertebrates. For this report, we assume that all vertebrates are likewise
capable of experiencing pain.
2. Assuming similarities in pain between humans and animals 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. In a fashion similar to the emphasis stated in the sister Distress report,
the Three Rs (replacement, refinement and reduction) should be the standard
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 optimum 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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