4
Recognition and Assessment of Pain, Stress, and Distress

This chapter builds on the definitions provided in Chapter 1 and the discussions of pain and environmental sources of stress and distress in Chapters 2 and 3. It is intended to enable readers to recognize and assess pain, stress, and distress in laboratory animals for the purposes of developing therapeutic, environmental, and behavioral strategies for decreasing them and lessening their impact on experimental data. The recognition of pain, pain-induced distress, and non-pain-induced distress in animals is ethically necessary for proper clinical management of animals to ensure their well-being and to reduce research variability.

RECOGNITION AND ASSESSMENT OF PAIN

The responses of humans to potential or actual tissue damage are parts of a complex experience that has sensory qualities and motivational and emotional consequences. The nervous system encodes the sensory features of tissue-damaging stimuli, such as their quality, intensity, location, and duration. What we perceive results in behavioral and physiologic responses that are under the influence of emotional, motivational, and cognitive processes. Noxious or tissue-damaging stimuli are unpleasant and can evoke strong negative feelings that include memories of previous discomfort, cultural beliefs about pain, and our awareness that pain can imply serious harm to our body. Thus, as described in Chapters 1 and 2, a simplified view of the pain experience includes two major components: sensory and affective or emotional.



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Recognition and Alleviation of Pain and Distress in Laboratory Animals 4 Recognition and Assessment of Pain, Stress, and Distress This chapter builds on the definitions provided in Chapter 1 and the discussions of pain and environmental sources of stress and distress in Chapters 2 and 3. It is intended to enable readers to recognize and assess pain, stress, and distress in laboratory animals for the purposes of developing therapeutic, environmental, and behavioral strategies for decreasing them and lessening their impact on experimental data. The recognition of pain, pain-induced distress, and non-pain-induced distress in animals is ethically necessary for proper clinical management of animals to ensure their well-being and to reduce research variability. RECOGNITION AND ASSESSMENT OF PAIN The responses of humans to potential or actual tissue damage are parts of a complex experience that has sensory qualities and motivational and emotional consequences. The nervous system encodes the sensory features of tissue-damaging stimuli, such as their quality, intensity, location, and duration. What we perceive results in behavioral and physiologic responses that are under the influence of emotional, motivational, and cognitive processes. Noxious or tissue-damaging stimuli are unpleasant and can evoke strong negative feelings that include memories of previous discomfort, cultural beliefs about pain, and our awareness that pain can imply serious harm to our body. Thus, as described in Chapters 1 and 2, a simplified view of the pain experience includes two major components: sensory and affective or emotional.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals There should seldom be a question about the possibility that a laboratory animal is in pain, if basic principles are followed. U.S. Government Principles for Utilization and Care of Vertebrate Animals Used in Testing, Research, and Training (IRAC, 1985) states that "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." Experimentally induced pain is ordinarily predictable and either avoidable or relievable, according to the requirements of the research protocol. But unforeseen circumstances might place an animal in unexpected pain, and there must be ways to recognize it. In evaluating procedures that cause pain and distress, one should justify the procedures and their potential benefits to humans or animals. Societal concerns about the welfare of animals used for experimental purposes necessitate that standards be developed to take into account all relevant information, including scientific data and observations from both biologic and behavioral sources. Guidelines should be established that ensure relief from pain or distress in any study (Dubner, 1987; Montgomery, 1987). Pain, stress, and distress that are not produced specifically for study should be viewed as unnecessary, unwanted, data-compromising side effects (Amyx, 1987; Spinelli and Markowitz, 1987). It must be emphasized that stress can still affect experimental results even if an animal under stress seems to be adapting and is manifesting no maladaptive behavioral or physiologic signs. CLINICAL APPROACHES TO THE ASSESSMENT OF PAIN One of the more important responsibilities in the use of animals for biomedical research is to recognize clinical signs associated with pain. Without a knowledge of their normal and abnormal behavior and appearance, assessment of pain in animals is difficult, because animals are unable to communicate in ways in which they can be readily understood by people (Hughes and Lang, 1983; Soma, 1987). An important step in determining that an animal is in pain is the recognition of a departure from the animal's normal behavior and appearance (Morton and Griffiths, 1985; Dubner, 1987; Kitchen et al., 1987; Dresser, 1988). Well-being is usually associated with species-typical behavior and is used as a descriptor for healthy animals that are adapted to their environment (however, see Chapter 3). A well animal will play with its cagemate or handler, exhibit normal curiosity through explorations, keep itself well groomed, appear to be in good health, grow normally, and have normal reproduction. There are no generally accepted objective criteria for assessing the degree of pain that an animal is experiencing (Morton and Griffiths, 1985), and species vary widely in their response to pain. However, some behavioral signs are usually associated with pain (Table 4-1). Animals often communicate pain by their posture.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 4-1 Signs of Acute Paina Sign Explanation Guarding Attempting to protect, move away, or bite Vocalization Crying out when palpated or forced to use affected area Mutilation Licking, biting, scratching, shaking, or rubbing Restlessness Pacing, lying down and getting up, or shifting weight Sweating In species that sweat (horses) Recumbency Unusual length of time Depression Reluctance to move or difficulty in rising Abnormal appearance Head down, tucked abdomen, hunched, facial distortion, or pallor a Reprinted with permission from Soma, 1987. For example, an animal with abdominal pain might sit hunched up or hold a limb in an abnormal position. Occasionally, an animal in severe pain flails about, struggles, writhes, or has extensor rigidity. The affected area could be wet from repeated licking, and licking can progress to self-mutilation and chewing at the affected area. Wounds—self-inflicted, from other animals, or from surgery—can appear red and swollen. Assessment of signs of less severe pain involves at least some subjective interpretation and requires careful clinical observation and familiarity with animals' responses to similar situations (Soma, 1987). Animals in pain often display an appearance that is best described as an ''absence of normal behavior." Normal behavior can be characterized by various species-typical actions, such as cats' response to stroking, goats' head pushing, and pigs' or primates' general activity and vocalization. When an animal is in pain, these aspects of behavior can be strikingly absent; the animal might withdraw to the rear or corner of its cage or become aggressive and show signs of attack, or it might even become listless, inactive, or immobile (Flecknell, 1985). Assessing Vocalizations Vocalizations are natural reactions to pain in many animals and can be used as a guide to the degree of pain (Lefebvre and Carli, 1985; Cooper and Vierck, 1986). An animal might squeal, bark, or otherwise phonate when handled. Vocalization also includes groaning, grunting, whimpering, whining, and growling. Some noises indicative of pain are characteristic of a particular species; they are usually more than momentary and are often repeated. Vocalizations associated with pain can be used to measure an animal's reactivity to pain. Attempts to restrain pigs and nonhuman primates that are in pain can cause vocalizations, but these and

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Recognition and Alleviation of Pain and Distress in Laboratory Animals other animals also vocalize in association with feeding or the presence of a familiar person or strangers. In many species, vocalizations differ enough to convey specific meanings to careful observers. However, vocalizations are not definitive and reliable indicators of pain in some animals, and the absence of vocalization is not an invariable indicator of the absence of pain. Furthermore, many vocalizations of rodents, and possibly other animals, are at frequencies above the hearing range of people. Responses to Handling In any clinical examination of an animal, inspection and palpation are important. Before restraint is applied, an animal's posture or movement should be observed for evidence and localization of pain. Once the animal is restrained, a complete physical examination should be conducted (see Pare and Glavin, 1986, for a review of the stress caused by restraint). Knowledge of the animal's normal response to handling is desirable, because individual animals can respond differently. An animal in acute pain might have increased muscle tone, show reluctance to be handled, and guard the painful area. It might turn its head toward the source of pain when the area is touched, attempt to bite its handler, or cry out if the painful site is manipulated. Those signs can often be associated with activation of the sympathetic nervous system as manifested in increased heart and respiratory rate, dilated pupils, increased body temperature, sweating, and muscle tremors. Elicitation of a response associated with pain, such as movement of the head toward the area being palpated, might be necessary to show that the animal is in pain and to locate the painful area. Palpation of the abdomen often causes the muscles to tighten, and the animal might grunt, squeal, or attempt to bite. Acute Pain Acute pain plays a protective role: it warns about injury. Acute pain can be produced by transient stimuli, such as venipuncture or brief electric shock. Such pain produces a stress response, but usually does not lead to distress, because the pain is short-lived. In the face of such phasic tissue-damaging stimuli, animals are generally able to adapt their behavior and accept their discomfort. Acute pain also can result from an inflammatory process that originated in damaged tissue, surgery, traumatic injury, or exposure to metabolic, bacterial, or viral disease or toxins. Behavioral responses vary among breeds and species, but the classic signs of inflammation are universal: pain, edema, redness, increased temperature, and loss of function. Those signs are mediated by the release of chemical mediators, such as bradykinin, prostaglandins, leukotrienes, substance P, serotonin, and histamine (Chapter 2). The inflammatory process produces increased neural activity originating in the response of receptors to tissue-damaging stimuli.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Chronic Pain Chronic or persistent pain is different from acute pain and can be harder to recognize, because its onset is slow, its intensity is likely not constant, it is not necessarily associated with an obvious pathologic condition, and it usually does not serve any vital protective function. It is also more likely to lead to distress and maladaptive behavior. Animals in chronic pain can be divided into three broad categories: those with a known pathologic condition (e.g., arthritis, cancer, or injury), those in which an organic cause of the pain can be inferred from the results of the clinical examination and history (e.g., pain of musculoskeletal origin, peripheral nerve damage, or disease of the central nervous system), and those with signs that resemble signs in one of the other categories but without obvious cause. Animals in all three categories can exhibit signs of psychologic or psychosocial dysfunction. Some signs are likely to be common to chronic pain of any origin. They include decreased appetite, weight loss, reduced activity, sleep loss, irritability, and decreased mating and reproductive performance (Soma, 1987). Alterations in urinary and bowel activities and lack of grooming are signs often associated with chronic pain, and tearing and lacrimal accumulations around the eyes should be noted. Animals whose pain is chronic or that are moribund might exhibit reduced body temperature, a weak, shallow pulse, and depressed respiration—signs of a poor prognosis. Chronic pain of the musculoskeletal system is fairly easy to recognize because of lameness or reluctance to move. Some chronic conditions can cause an animal to harm itself; e.g., licking can progress to rubbing, chewing, or scratching, and occasionally self-injury becomes so severe as to mask the cause. Even if a cause is identified and corrected, maladaptive behaviors might persist and require careful diagnosis and treatment (Chapter 5). Classification of Procedures Likely to Cause Pain As with all surgical procedures, appropriate anesthesia should be used to render the animal insensitive to pain. However, the postsurgical period is most likely to be associated with pain; pain should be expected, and appropriate use of analgesics should be planned and described in the research protocol. Table 4-2 lists signs, degrees, and durations of pain associated with various classes of surgical procedures. The Committee on Animal Research of the New York Academy of Sciences (NYAS, 1988) has developed guidelines for research that uses animals. Table 4-3 lists examples of experiments of various types and some ethical considerations relevant to them. The guidelines are of value to scientists who are designing experiments and to institutional animal care and use committee (IACUC) members who are trying to identify painful and distressful procedures.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 4-2 Signs, Degree, and Length of Surgically Produced Paina Surgical Site Signs of Pain Degree of Pain Length of Pain Head, eye, ear, mouth Attempts to rub or scratch, self-mutilation, shaking, reluctance to eat, drink, or swallow, reluctance to move Moderate to high Intermittent to continual Rectal area Rubbing, licking, biting, abnormal bowel movement or excretory behavior Moderate to high Intermittent to continual Bones Reluctance to move, lameness, abnormal posture, guarding, licking, self-mutilation Moderate to high: upper part of axial skeleton (humerus, femur) especially painful Intermittent Abdomen Abnormal posture (hunched), anorexia, guarding Not obvious to moderate Short Thorax Reluctance to move, respiratory changes (rapid, shallow), depression Sternal approach, high; lateral approach, slight to moderate Continual Spine, cervical Abnormal posture of head and neck, reluctance to move, abnormal gait—"walking on eggs" Moderate to severe Continual Spine, thoracic or lumbar Few signs, often moving immediately Slight Short a Based on observations of dogs. SPECIES-TYPICAL SIGNS Species-typical signs should be taken into account in an assessment of pain. Experience suggests strongly that some signs are often associated with pain. No sign, however, can by itself regarded as diagnostic of pain, because similar signs occur in conditions in which pain is unlikely. Signs are not all present at one time, and those present on one examination can change by the next. Signs of pain should therefore be considered as a complex group and evaluated together.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 4-3 Guide to Types of Experiments That Are Considered Painful or Stressfula Types of Experiments Examples of Procedures Special Ethical Considerations A. Experiments on unanesthetized animals not expected to have pain and/or distress. Naturalistic observations of behavior (e.g., in the field, in nonextreme environments); social interactions and noninvasive procedures; EEG, EKG, EMG in trained animals; minimal nutritional modifications; conditioning or training procedures using appetitive motivation; habituation to nonaversive stimuli Appropriate species, strain; health status and history; duration of procedure; disruption of behavioral pattern of species; disruption of other coexisting species during field studies B. Experiments on unanesthetized animals which may be expected to have no more than minimal pain and/or distress. Procedures that are expected to be no more painful or distressful than administration of a medication or anesthetic agent: experiments involving minimal physical restraint related to physical examination, EEG, EKG, EMG in untrained animals; minimal deprivation; learning or conditioning with aversive stimulation of short duration or nontraumatic magnitude (e.g., mild shock, corneal air puff); injections/collections of blood samples or other body fluids; pain titration; dietary feeding; models of minimal disease; tumor growth not affecting normal function; product safety testing; short- and long-term studies of effects of chemical or other agents known not to have significant noxious, addicting, or intoxicating effects and measured noninvasively; pithing of frogs As in Type A; duration of procedure; duration or restraint and/or deprivation; pithing should be carried out with the animal immobilized and wherever feasible at a temperature no greater than 40°F.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Types of Experiments Examples of Procedures Special Ethical Considerations C. Experimental use of animals after they have been painlessly killed. Removal of tissue or organs for study Method of euthanasia D. Acute experiments on anesthetized animals which will not be permitted to recover from anesthesia. Physiological and neurophysiological studies; studies with chemical agents (e.g., CNS stimulants and depressants); nonsurvival surgical procedures Mode and effectiveness of anesthesia; if neuromuscular (paralytic) agents are used in combination with anesthesia, appropriate personnel and equipment must be available to assure that the animal is ventilated and remains anesthetized. E. Experimental procedures, including surgical procedures, carried out under general or regional anesthesia with intent of recovery. Recovery period with the animal's adaptation to the experimental intervention is considered an integral part of the experimental protocol, and no serious modifications to the animal's behavior, feeding, or ambulatory patterns are anticipated. No more than minimal to moderate pain and/or distress is anticipated which can be altered when necessary by the administration of appropriate analgesic and sedative drugs and proper veterinary care. Biopsies, catheterizations, implantation of biomedical material, injection of substances to produce inflammatory reaction (e.g., lactic acid, colchicine); abdominal, thoracic or peripheral surgery (e.g., for implantation of chronic monitoring or support devices and for modification of internal organs); peripheral and central nervous system lesions and implants; pituitary removal; direct treatment, transfer, or sampling of fetuses or embryos in utero As in Types A–D; appropriate facilities, aseptic technique and surgical procedures for the species involved; postoperative nursing care which also includes maintenance of body temperature, hydration, and control of pain and infection; duration of procedure; duration of postoperative pain and distress and consideration of use of analgesics; approval of full IACUC is necessary. F. Experimental procedures, including surgical procedures as in E above, but where study is a progression of surgical or preoperative period and where surgical preparation may be a component of the overall study. The observation and study period after the intervention may produce more than moderate pain, distress, or illness and/or significantly impair the ability of the animal to function in its environment. Shock; burn and organ transplant and rejection studies; severe trauma; extensive lesions or ablations As in Type E and additional requirements for analgesia and sedation during remainder of procedure if necessary; approval of full IACUC is required.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Types of Experiments Examples of Procedures Special Ethical Considerations G. Experiments on unanesthetized animals which may be expected to have more than minimal pain and/or distress and/or where death is anticipated. Administration of compounds resulting in damage to vital tissue or serious alteration of function; tests of drug efficacy to control severe pain; models of severe disease; severe trauma, injury, or shock; prolonged intense aversive stimulation; extensive tumor growth affecting normal function; studies which do not permit the animal to control the amount of intense painful stimuli it will receive or where drugs will not be administered to modify severe pain or distress; highly invasive studies of major sensory pathways; shortand long-term studies of the effects of chemical or other agents but known or expected to have significant noxious, addicting, or intoxicating effects As in Type F and additional requirements for analgesia and sedation during remainder of procedure if necessary; specific justification is required to approve traumatic studies without proper anesthesia, analgesia, or sedation or with the nonsurgical use of neuromuscular (paralytic) agents without sedation or anesthesia; such studies must be conducted with special consideration for comfort of the animal and number of animals used; the investigator must specify the endpoint of the experiment as well as alternative situations in which termination of the experiment would be mandatory to avoid prolongation of suffering; such specification may be based on pathologic, physiologic, or behavioral observations; these procedures must not be carried out without approval of the full IACUC. a Reprinted with permission from New York Academy of Sciences (NYAS, 1988).

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Similarities and Differences in Signs Among Humans and Animals Humans often describe pain as sharp, dull, pricking, burning, or itching. Animals cannot relate such descriptions, so pain has to be assessed by observing their behavioral or physiologic reactions. Although many similarities between humans and animals can be used for pain detection (Vierck, 1976; Vierck et al., 1983; Zimmermann, 1984; Kitchell and Johnson, 1985), marked differences in pain tolerance must be kept in mind. The anthropomorphizing of pain perception should be tempered by the recognition of the many differences between humans and animals. Pain thresholds are remarkably similar among all species and breeds of animals, but the perceived intensity and tolerance of pain vary among individual animals and in the same animal under different circumstances. Many factors—including strain, species, experience, age, health, and stress—affect pain tolerance (Wright et al., 1985; Breazile, 1987). Young animals might have a lower tolerance of acute pain than do older ones. A systemically ill animal might be less tolerant of pain than a healthy one, but a moribund or severely ill animal might be nonresponsive albeit in distress. Some marked differences in pain responses between humans and animals are related to the site of pain. Abdominal surgery is thought to be less painful in four-legged animals than in humans, because humans use their abdominal muscles to a much greater extent in maintaining posture and for walking. A median sternotomy could be classified as producing low to moderate pain in humans and much pain in animals, probably because animals use their front limbs in walking and movement of sternal edges after the sternum has been surgically separated would be slight but painful during walking. A lateral thoracotomy is likely to be accompanied by less pain in animals than in humans, because respiration is more abdominal in animals and more thoracic in humans. Such differences account at least in part for the wide variety of signs seen in response to surgery (Soma, 1987). Nonhuman Primates Nonhuman primates show remarkably little reaction to surgical procedures or to injury, especially in the presence of humans, and might look well until they are gravely ill or in severe pain. Viewing an animal from a distance or by video could aid in detecting subtle clinical changes. Loud and persistent vocalization is an occasional but unreliable expression of pain; it is more likely to signify alarm or anger. Therefore, it should be recognized that a nonhuman primate that appears sick is likely to be critically ill and might require rapid attention. A nonhuman primate in pain has a general appearance of misery and dejection. It might huddle in a crouched posture with its arms across its chest and its head forward with a "sad" facial expression or a grimace and glassy eyes. It might moan or scream, avoid its companions, and stop grooming. A monkey in pain can also attract

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Recognition and Alleviation of Pain and Distress in Laboratory Animals altered attention from its cagemates; this can vary from a lack of social grooming to attack. Acute abdominal pain can be shown by facial contortions, clenching of teeth, restlessness, and shaking accompanied by grunts and moans. Food and water are usually refused. Dogs Dogs in pain generally appear less alert and quieter than normal and have stiff body movements and an unwillingness to move. A dog in severe pain might lie still or adopt an abnormal posture to minimize its discomfort. In less severe pain, dogs can appear restless and more alert. There can be inappetence, shivering, and increased respiration with panting. Spontaneous barking is unlikely. They are more likely to whimper or howl, especially if unattended, and might growl without apparent provocation. Small breeds are generally more reactive to environmental changes than large dogs. Dogs can bite, scratch, or guard painful regions. When handled, they might be abnormally apprehensive or aggressive. Cats Cats are less reactive to environmental changes than dogs. A cat in pain is generally quiet and has an apprehensive facial expression, and its forehead might appear creased. It is inappetent and might cry, yowl, growl, or hiss if approached or made to move. It tends to hide or to separate itself from other cats. Its posture becomes stiff and abnormal, varying with the site of pain. If the pain is in its head or ears, it might tilt its head toward the affected side. A cat with generalized pain in both the thorax and abdomen might be crouched or hunched. If the pain is only thoracic, the head, neck, and body might be extended. A cat with abdominal or back pain might stand or lie on its side with its back arched or walk with a stilted gait. Incessant licking is sometimes associated with localized pain. Pain in one limb usually results in limping or holding up of the affected limb with no attempt to use it. Cats in severe or chronic pain look ungroomed and behave markedly differently from normal. Touching or palpation of a painful area might produce an immediate violent reaction and an attempt to escape. A general lack of well-being is an important indication of pain in cats. Rabbits Rabbits in pain can appear apprehensive, anxious, dull, or inactive and assume a hunched appearance, attempt to hide, and squeal or cry. But sometimes they show aggressive behavior with increased activity and excessive scratching and licking. Reactions to handling are exaggerated, and acute pain might result in vocalization. With abdominal pain, they sometimes grind their teeth and salivate excessively. Their respiratory rate can be increased, and they can be inappetent.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Rabbits in distress might cannibalize their young and tend to be more susceptible to the tonic immobility reflex, a phenomenon that is thought to block pain in prey species (see Chapter 5). Laboratory Rodents Pain in rodents usually results in decreased activity, piloerection, and an ungroomed appearance. There can be excessive licking and scratching, which can progress to self-mutilation. They might adopt an abnormal stance or a hunched posture. Respiration can be rapid and shallow with grunting or chattering on expiration. Pupils might be dilated. In albinos, porphyrin secretion ("red tears") can be seen around the eyes and nose. Rats and mice in acute pain might vocalize and become unusually aggressive when handled. Their squeal can be at an unusually high pitch or at a frequency above human hearing. Inappetence or a change in feeding activity can be noted. They might eat bedding or their offspring. If they are housed with others, the normal group behavior or grooming might change. They might separate from the rest of the animals in the cage and attempt to hide. Normal guinea pigs will stampede and squeal when frightened, when attempts are made to handle them, or when strangers are in the room, but sick guinea pigs and those in pain will usually remain quiet. Other behaviors of guinea pigs in pain are similar to those of rats and mice. Horses Horses in acute pain show reluctance to be handled, and their other responses are varied: periods of restlessness, interrupted feeding with food held in the mouth uneaten, anxious appearance with dilated pupils and glassy eyes, increased respiration and pulse rate with flared nostrils, profuse sweating, and a rigid stance. In prolonged pain, their behavior might change from restlessness to depression with head lowered. In pain associated with skeletal damage, there is reluctance to move; limbs might be held in unusual positions, and the head and neck in a fixed position. Horses with abdominal pain might look at, bite, or kick their abdomen; get up and lie down frequently; walk in circles; and sweat, roll, and injure themselves as a result of these activities, with bruising especially around the eyes. That state can progress and last for several hours. When near collapse, they might quietly stand rigid and unmoving, but with signs of deteriorating circulatory status, such as mucosal cyanosis and prolonged capillary filling time. Cattle Cattle in pain often appear dull and depressed, hold their heads low, and show little interest in their surroundings. There is inappetence, weight loss, and, in

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Recognition and Alleviation of Pain and Distress in Laboratory Animals milking cows, a sudden decrease in milk yield. Severe pain often results in rapid, shallow respiration. On handling, they might react violently or adopt a rigid posture designed to immobilize the painful region. Grunting and grinding of teeth might be heard. Localized pain might be associated with persistent licking or kicking at the offending area and, when the pain is severe, with bellowing. Generally, signs of abdominal pain are similar to those in horses, but less marked. Rigid posture can lead to a lack of grooming because of an unwillingness to turn the neck. In acute abdominal conditions, such as intestinal strangulation, cattle adopt a characteristic stance with one hind foot placed directly in front of the other. Sheep and Goats In general, signs of pain in sheep and goats are similar to those in cattle, but sheep, in particular, tolerate severe injury without overt signs of pain or distress. Changes in posture and movement are often apparent, and a change in facial expression might be observed. There is a general reluctance to move. Goats are more likely than cattle to vocalize in response to pain. Grinding of teeth and grunting are also heard. After castration or tail docking, lambs might show signs of pain by standing and lying repeatedly, wagging their tails, occasionally bleating, and displaying neck extension, dorsal lip curling, kicking, rolling, and hyperventilation. Pigs Pigs in pain might show changes in social behavior, gait, and posture and an absence of bed-making. Pigs normally squeal and attempt to escape when handled, and pain can accentuate these reactions. Adults might become aggressive. Squealing is also characteristic when painful areas are palpated. Pigs in pain often are unwilling to move and might hide in bedding if possible. Birds Birds in pain can show escape reactions, vocalization, and excessive movement. Small species struggle less and emit fewer distress calls than large species. Head movements increase in extent and frequency. There can be an increase in heart and respiratory rates. Prolonged pain results in inappetence, inactivity, and a drooping, miserable appearance. The eyes might be partially closed, the wings held flat against the body, and the neck retracted. When a bird is handled, its escape reaction might be replaced by tonic immobility (see Chapter 5). Birds with limb pain avoid use of the affected limb and ''guard" it from extension. Reptiles Acute pain in reptiles can be characterized by flinching and muscle contractions. There might be aversive movements away from the unpleasant stimulus and

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Recognition and Alleviation of Pain and Distress in Laboratory Animals attempts to bite. More chronic and persistent pain might be associated with anorexia, lethargy, and weight loss, although it is difficult to associate any of these signs of lack of well-being specifically with pain. Fish It is difficult to determine the nature of the response to pain in fish, and we cannot tell whether the experience is similar to that in mammals (Arena and Richardson, 1990). Fish exhibit a pronounced initial response to injuries or to contact with irritants, but their response to chronic stimuli might be small or absent. Fish with severe wounds, which would cause immobility in a mammal, often appear to behave normally and even resume feeding. Fish react to noxious stimuli, such as puncture with a hypodermic needle, with strong muscular movements. When exposed to a noxious environment, such as an acidic solution, they show abnormal swimming behavior and attempt to jump from the water, their coloring becomes darker, and their opercular movements become more rapid. Such effects indicate some, perhaps considerable, distress, but it is not possible to describe the distress unequivocally as pain-induced. RECOGNITION AND ASSESSMENT OF STRESS AND DISTRESS The most potent sources of stress in captive environments, other than pain, are likely to fall within the six ecologic dimensions described in Chapter 3; some of the most common are summarized in Table 4-4. Some useful information for a given species on conditions in the captive environment that are likely to be stressful can be gleaned from descriptions of the behavior of the species in its natural habitat, but such information is no substitute for data based on experiments carefully conducted in the captive environment. In assessing stress and distress in an animal, one should first determine whether the source is pain (acute or chronic) or other factors. That might be evident from the animal's recent history. If the animal is in pain, it might also be in distress. Or some unrelated, nonpain stressor could be producing distress or potentiating the pain. If it is determined that a stressor other than pain is contributing to the animal's distress, its nature and source should be determined (Chapter 3). Distress caused by pain alone generally abates when the pain is relieved. But non-pain-induced distress should be addressed as a problem separate and distinct from pain and usually requires a nonpharmacologic (environmental) approach. As indicated earlier, stress and distress are complex syndromes that are difficult to define and even harder to interpret and recognize (Wright et al., 1985). The recognition and assessment of stress and distress and the identification of events that induce them present many problems not often apparent to the untrained observer (Crane, 1987). The syndromes are among the physiologically most complex and psychologically compelling experiences that a human, and presum-

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Recognition and Alleviation of Pain and Distress in Laboratory Animals TABLE 4-4 Potential Causes of Stress in Laboratory Animalsa Husbandry Practices • Inappropriate or variable temperature, humidity, ventilation, or illumination • Inappropriate cage or enclosure size • Noise • Too infrequent change in bedding or removal of waste • Stale food or dirty water • Denial of positive social stimulation • Maternal deprivation • Social intimidation or abuse by companions • Unprofessional behaviors or practices Experimental Design • Food and water deprivation • Inadequate caging • Poor or inappropriate technique • Failure to adapt or handle animals • Restraint • Social deprivation • Frequent changes in procedures or personnel a This table is not meant to be all-inclusive and excludes situations in which pain-induced stress can arise (e.g., postsurgical recovery). It is assumed that animals are otherwise physically healthy. ably an animal, can have. It is important that we develop the ability to recognize and deal with them in animals (Steffey, 1983). Just how a particular species will respond and cope with stimuli that can induce stress is difficult to predict. There can be a great deal of interspecies and individual variability, so one animal might respond quite differently from another (Vierck, 1976). That variability can be decreased in some common laboratory animals, such as mice and rats, in which generations of breeding have tended to produce animals that respond to stressful stimuli relatively uniformly (Hughes and Lang, 1983). But variability is accentuated in highly outbred populations, such as dogs and cats, or in such a highly diverse order as nonhuman primates. Some breeds of dogs, such as hounds, are noted for their hardiness and stoic demeanor and might be less likely to show stress in response to the same stimuli that cause sharp reactions in other breeds, such as toy poodles. DIAGNOSIS OF STRESS AND DISTRESS The following discussion approaches the diagnosis of pain-induced and non-pain-induced stress and distress from a problem-oriented perspective. Table 4-5 lists common behaviors and physiologic and biochemical characteristics, changes in which can indicate stress or changes in well-being.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Assessing an Animal's Records The first direct information about an animal should be readily apparent from examination of its cage card and clinical record. This information should include species and breed, date the animal was received, genetic origin and vendor, age, sex, population density in the cage, previous experimentation or illness, and reproductive activity or status (Spinelli and Markowitz, 1987). Surgical procedures—including the use of anesthetics and analgesics and presurgical and postsurgical care—should be included in the record. Food intake should be examined, not only for amount, but for pattern of feeding or waste. Waste of food or failure to eat can indicate illness, spilling, lack of palatability, spoiling, or oral lesions. Assessing the Environment A scheme should be developed for assessing each animal in a logical, organized fashion. Initial assessment should be carried out at a distance (Morton and Griffiths, 1985; Wright et al., 1985). The animal should be observed for its appearance and behavior before it is disturbed. On entry into the animal room, the assessor should be aware of the animal's environment. The room temperature, humidity, ventilation, odors, and indication of contamination by vermin should all be considered. A well-trained clinician or technician can often "feel" abnormal environmental situations. If the assessor is unsure about the animal's well-being, expert advice should be sought. TABLE 4-5 Some Behavioral, Physiologic, and Biochemical Indicators of Well-Being Behavioral Physiologic Biochemical Grooming Temperature Corticosteroids Appetite Pulse Catecholamines Activity Respiration Thyroxin Aggression Weight loss Prolactin Facial expression Blood-cell count β-Endorphin Vocalization Blood-cell structure ACTH Appearance Cardiac output Glucagon Posture Blood flow Insulin Response to handling   Vasopressin     Substance P a Departures from normal behaviors and characteristics are suggestive of changes in well- being. A knowledge of species-typical and individual-specific behaviors and clinical values is essential.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Stress and Distress Caused by Lack of Environmental Stimuli Most attention to animal stress focuses on techniques that involve obviously aversive stimuli. In human medicine, however, it is known that lack of external stimuli can cause psychiatric disorders. Researchers have demonstrated that laboratory animals sometimes develop severe signs of distress from a lack of essential stimulation. (This topic is discussed in greater detail in Chapter 3.) The recent focus on the "psychologic well-being" of nonhuman primates has raised the issue repeatedly, and many have proposed to improve psychologic well-being with enrichment devices or programs. Although nonhuman primates have received nearly all the attention, it should be asked whether other laboratory animals are in distress as a result of environmental monotony in standard housing or lack of other unidentified components of the environment. Understimulated animals of many species show abnormal behavior patterns (stereotypes and displacement behaviors) that are largely absent in enriched environments (Wemelsfelder, 1990). Animals in unenriched environments are also more passive, and their behaviors might be less diverse than those of animals in more stimulating surroundings. Assessing a Species and an Individual Animal When stress is present, the first change most likely to be reported is a change in an animal's activity pattern. Behavior can range from inactivity to hyperactivity and from adaptive to maladaptive, depending on the source and severity of the stressor and the species. Changes can be seen in sleep and eating behavior. The animal might be nonresponsive, listless, lethargic, and depressed, or it might be unusually restless, excitable, anxious, apprehensive, hypersensitive, or aggressive. It might constantly move about the enclosure or repeatedly stand and lie down. As one approaches a normal animal's cage, it should respond in a usual and predictable manner that enables assessment of its gait, inquisitiveness, vocalization, and posture. An animal in stress that previously would have investigated a new visitor to the room or a change in the environment might now fail to do so or attempt to escape. Nocturnal species, which during daylight hours exhibit very little species-typical behavior other than sleeping, should be observed at times that coincide with their active period, e.g., early in the morning, when the animals are still awake and moving about. During initial observations, respiratory and activity patterns should be assessed without investigator-produced stimulation or the escape behaviors that are often associated with capturing and handling. Assessing Behavior The definition and measurement of distress are in their infancy. Few attempts have been made to place the assessment of distress on a scientific and systematic

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Recognition and Alleviation of Pain and Distress in Laboratory Animals basis. The transition between atypical behaviors expressed as a homeostatic process of adapting to stress and the maladaptive behaviors expressed as distress might not always be clearly defined. Atypical behaviors can be developed by an animal to cope with the constraints of a laboratory environment or experimental conditions and might indicate that it is in acute stress. However, assessment of the meaning of an atypical behavior requires the most diligent attention and professional judgment. Although an animal that shows such behavior is not necessarily in distress, the potential transition period between acute stress and distress should be of concern to investigators and animal staffs. The duration of atypical behaviors and the type of behaviors involved should constitute a warning that an animal needs attention; serious attempts must be made to alleviate the potential for distress. Distinctive species-typical responses to pain and to fear are shown by many species and are usually easy to recognize. A limitation on the use of these species-typical indicators is that the conditions that elicit pain and fear make up a relatively narrow and specific subset of the total range of captivity situations that are likely to cause distress. A situation in which an animal is unable to perform all the normal or instinctive behaviors of its species should not always be considered as causing stress or even distress. As a blanket prescription, this has obvious shortcomings. The assumption is that a monkey that never screams in fear, a hen that is unable to dust bathe, a cat that is unable to capture live prey, or a rabbit or rat that is unable to burrow is distressed. The committee believes that an expression of species-typical behavior is preferable, but that a state of well-being can exist even if species-typical behaviors are not all manifest. There is no evidence that all normal activities of a species are based on specific behavioral needs or motives whose blocking causes distress. As indicated in Chapters 1 and 3, an animal that shows behaviors that are abnormal or atypical for its species should not always be considered distressed. To be sure, some behaviors are clearly contrary to well-being and are undesirable on that basis alone; self-mutilation is an example. But animals might develop other atypical behaviors to cope with the constraints of their captive environment or to take advantage of the special opportunities it provides. Rather than being signs of distress, some atypical behaviors could be ways of adapting to stress and reducing or controlling distress. Another approach to the assessment of distress is based on the reasonable assumption that situations that act as rewards or provide positive reinforcement are pleasant for an animal, and situations that it will work to avoid are unpleasant or distressful (Dawkins, 1976, 1983). Dawkins (1990) suggests that distress occurs in situations in which captive animals "are prevented from doing something they are highly motivated to do." No criterion or method for the assessment of distress is without value, and none is free of limitations. The presence or absence of species-typical behaviors should certainly be considered, although it is not sufficient for evaluating distress. Maladaptive behavior is important, but should be interpreted cautiously. Behav-

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Recognition and Alleviation of Pain and Distress in Laboratory Animals ioral methods for measuring motivation, such as those proposed by Dawkins, have been available for many years and can play an important role in the assessment of distress, but they are not without problems. No measure of motivation provides an adequate index of its "strength" (e.g., Miller, 1956). Motivation, like stress, does not refer to a unitary process or single "drive" (e.g., Fentress, 1973; Mason and Capitanio, 1988). Furthermore, the relationship between the strength of motivational "demand" and distress is not obvious. It is at least conceivable that an animal can be highly motivated to achieve a particular "commodity" and yet show no signs of distress or other untoward effects when this commodity is not available. Behavioral changes, however, are the earliest signs of stress or distress that most animal care staff and researchers are likely to confront. Skilled observers who know the behavior of a particular species or strain of animal and of the individual animals under their care could provide a reliable assessment of the state of the animals. That reliability is seriously compromised when few animal care staff and researchers are afforded the time or training necessary for them to become skilled observers. Barclay et al. (1988) argue that departures from normal behavior in rats and mice can be produced by relatively minor procedures, but that some behaviors—such as feeding, drinking, and sleeping—have too high a priority to the animal to be used as baseline indexes for relatively minor stress, because they are not easily perturbed. Barclay et al. assume, from analogy and intuition, that change in behavior is related directly to severity of pain and stress. Whether or not that is true, a change in behavior should alert human observers. For example, Antin et al. (1975) report that rats engage in a specific and predictable pattern of postprandial grooming behavior; a persistent change in this pattern clearly suggests that something is amiss that involves the animal's well-being. PHYSIOLOGIC AND BIOCHEMICAL INDICATORS OF PAIN AND STRESS Although it used to be thought that the response to stress was an invariant physiologic reaction, recent studies support the idea that the stress response is not invariant, but depends on an integrated activation of various neural and endocrine factors. A variety of physiologic and biochemical characteristics change when an animal is in pain, stress, or distress (Dawkins, 1990), although many also change with the onset of general arousal, as in play or sexual excitement (see also Chapter 3). The relative importance of those factors depends on the type of stressor—physical (e.g., tissue damage or immobilization), physiologic (e.g., exercise), or psychologic (e.g., altered environment)—and on its magnitude, frequency, and timing. An animal can either habituate and adapt to a chronic stressor or develop maladaptive behavior that leads to distress. Regardless of whether the animal has habituated to a chronic stressor or shows maladaptive behavior, additional novel stimuli can evoke or increase the intensity of a stress response. Other factors that

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Recognition and Alleviation of Pain and Distress in Laboratory Animals can influence the stress response include age, sex, physical fitness, experience, disease, and medication. Neural and endocrine substances are released in response to a stressor and play an important role in the initiation and coordination of the behavioral, cardiovascular, and immunologic responses to stress. The site of origin of those substances influences the physiologic stress response, which has two categories of duration: a rapid increase in circulating neurally derived substances with a short duration of action and a slower increase in endocrine-derived substances with a longer duration of action. Acute stress—surgery, postoperative pain, burns, anesthesia, cardiac arrest, exercise, and sometimes headache—results in the hypothalamic secretion of corticotropin-releasing factor (CRF). CRF then stimulates the pituitary to cosecrete adrenocorticotropic hormone (ACTH), which promptly stimulates the release of corticosteroids from the adrenal cortex and the opioid peptides β-lipotropin and β-endorphin. In humans, plasma concentrations of these hormones can increase by a factor of 2–5 during stress. The roles of β-lipotropin and β-endorphin in the stress response are poorly understood. β-Endorphin might play a role in pain modulation (Hargreaves et al., 1983, 1987; Pickar et al., 1983; Szyfelbein et al., 1985). A potential target of circulating β-endorphin could be inflamed tissue (Joris et al., 1987). The major example of neurally derived responses to acute stress is the activation of the sympathoadrenal system. The pituitary-adrenal axis responds with a prompt increase in catecholamines from the adrenal medulla, which is usually followed by an increase in corticosteroids. Plasma concentrations of catecholamines increase considerably after surgery or postoperative pain, because of increased circulating concentrations of epinephrine, primarily from the adrenal medulla, and norepinephrine, primarily from sympathetic nerve terminal activation (Goldstein, 1987). Those different components of the sympathoadrenal system can be regulated separately. The sympathoneural release of norepinephrine evokes a regionally selective effect, whereas the adrenal medulla produces an increase in systemic plasma concentrations of epinephrine. The physiologic consequences of activation of those systems include increased cardiac output, increased skeletal muscle blood flow, cutaneous vasoconstriction, reduced gut motility, and increased glucose availability. Hormonally derived responses to stress result from changes in concentrations of growth hormone, prolactin, glucagon, insulin, vasopressin, neuropeptides, and other hormones. Acute stress causes increased secretion of growth hormone and prolactin, both from the anterior pituitary. Prolactin participates in metabolic processes and might modify nociception, but concentrations remain in the normal range in the presence of chronic stress. The significance of the short-lasting growth hormone increase is not known. Glucagon increases in response to acute stress, and insulin decreases. The decrease in insulin—in combination with increased prolactin, growth hormone, glucagon, and epinephrine—contributes to the development of hyperglycemia.

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Recognition and Alleviation of Pain and Distress in Laboratory Animals Surgery, burns, and migraine headaches in people can evoke vasopressin release from nerve endings in the posterior pituitary. Circulating vasopressin can influence vascular tone and diuresis and might also modulate nociception (Berson et al., 1983). Surgical trauma leads to increases in circulating neuropeptides, such as substance P and calcitonin gene-related peptide, possibly from unmyelinated nerve fibers activated at the site of inflammation and injury. Those neuropeptides are important in plasma extravasation and in the release of histamine from mast cells and serotonin from platelets. Their physiologic role after circulation in plasma is unknown. Responses to persistent stressors, such as chronic stress or distress, are more variable than responses to acute stress; catecholamines might return to normal values, and corticosteroid concentrations might be increased, unchanged, or decreased. Thus, changes in concentrations of pituitary and adrenal hormones can be used as markers of acute stress, but their usefulness in the recognition of chronic stress or distress is questionable at best. Biochemical markers are not unequivocal indicators of either pain or distress, but, when used in conjunction with behavioral and environmental data, they can reinforce a diagnosis. An acute stressor can produce a transient but important change in some characteristics; a chronic stressor can lead to the establishment of a new steady state of plasma hormones and heart function that might not be very different from the original. Different animals can respond to a given event differently, sometimes because of a lack of opportunity to habituate or acclimate. Care should be exercised when the effects of stress or distress could interfere with experimental results, because laboratory facilities and husbandry procedures can have different effects on individual animals. PHARMACOLOGIC ASSESSMENT OF DISTRESS It is not always possible to use behavioral or physiologic measures to distinguish between pain-induced distress and distress induced by other stimuli, but the use of pharmacologic techniques offers some interesting options in this regard. If an animal's behavior returns to normal after the administration of an analgesic, but not after administration of an anxiolytic, one would be justified in concluding that the distress was caused by a painful stimulus. One could similarly find that the distress was induced by fear or anxiety, rather than pain. Other drug-based approaches that raise some intriguing questions about animal distress include the recent investigations of the effects of opioid receptor antagonists on stereotypic behaviors in animals. Stereotypic behavior in a confined animal is sometimes considered to be a sign of distress. It is interesting that antagonists reduce or eliminate bar-chewing in confined pigs, crib-biting in stabled horses, and lick granulomas in dogs (Cronin et al., 1986; Dodman et al., 1987; White, 1990).